General Risk: There are many types of PAGE but they all involve handling monomer Acrylamide which is neurotoxic and can be absorbed through the skin. Even after polymerization there may be some free monomer present.
CONSEQUENTLY ALWAYS WEAR GLOVES WHEN HANDLING REAGENTS OR ELECTROPHORESIS GELS.
The following hazardous chemicals are used in the procedures below:-
Acrylamide - Toxic
Ammonium Persulphate - Harmful
Bisacrylamide - Harmful
Hydrochloric Acid (2M) - Corrosive
Mercaptoethanol - Harmful
Sodium Dodecyl Sulphate - Harmful
TEMED - Corrosive
General Protein Separation Method - Laemmli System
B. Non-Dissociating Discontinuous Buffers
C. General Purpose PAGE Reagents
E. Non-Dissociating Discontinuous Gel Preparation
F. General Preparation of Gels
G. Preparation Of Sample For SDS Electrophoresis
H. Preparation Of Sample For Non-Dissociating Electrophoresis
Low Molecular Weight Protein Separation
Coomassie Blue Detection of Proteins
A: Standard Method
B: Simple single step method
C: Simple single step method using Coomassie G (dye-binding Protein assay reagent)
Semi-dry electrophoretic transfer cell
Detection of Bands using the ECL method
Drying of Polyacrylamide Gel Slabs
i: Stacking Gel Buffer: 0.5 M Tris-HCL (pH 6.8)
6 g of Tris is dissolved in 40 ml of water. Titrate with 2M Hydrochloric Acid (approximately 24 ml) to pH 6.8. Make up to 100ml with water.
Risk Assessment
Preparation of Reagent - Can be prepared on the open bench. 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - There is no apparent hazard associated with this reagent
Spillage - Mop up with paper towels and place in yellow bag .
Disposal - The reagent can be flushed down the sink with excess water .
ii: Resolving Gel Buffer: 3.0 M Tris-HCl (pH 8.8)
Tris 36.3g HCl (2M) 24 ml Water to 100ml
Risk Assessment
Preparation of Reagent - Can be prepared on the open bench. 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - There is no apparent hazard associated with this reagent
Spillage - Mop up with paper towels and place in yellow bag .
Disposal - The reagent can be flushed down the sink with excess water .
iii: Stock Reservoir Buffer: 0.25M Tris, 1.92M Glycine, 1% SDS (pH 8.3)
Tris 30.3g Glycine 144g Sodium Dodecyl Sulphate (SDS) 10g Water to 1000 ml Dilute 1 in 10 before use
Risk Assessment
Preparation of Reagent - Sodium Dodecyl Sulphate generates irritant dust when being weighed out. Weigh out in fume cupboard wearing face mask. Add it carefully to the other components (preferably after addition of water).
Handling of Reagent - Little apparent hazard associated with this reagent
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
i: Stacking Gel Buffer: O.5 M Tris-HCL (pH 6.8)
6 g of Tris is dissolved in 40 ml of water. Titrate with 2M Hydrochloric Acid (approximately 24 ml) to pH 6.8. Make up to 100ml with water.
Risk Assessment
Preparation of Reagent - Can be prepared on the open bench. 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - There is no apparent hazard associated with this reagent.
Spillage - Mop up with paper towels and place in yellow bag.
Disposal - The reagent can be flushed down the sink with excess water.
ii: Resolving Gel Buffer: 3.0 M Tris-HCl (pH 8.8)
Tris 36.3g HCl (2M) 24 ml Make up to 100 ml with water.
Risk Assessment
Preparation of Reagent - Can be prepared on the open bench. 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - There is no apparent hazard associated with this reagent
Spillage - Mop up with paper towels and place in yellow bag .
Disposal - The reagent can be flushed down the sink with excess water .
iii: Stock Reservoir Buffer: 0.25M Tris, 1.92M Glycine (pH 8.3)
Tris 30.3g Glycine 144g Water to 1000 ml Dilute 1 in 10 before use
Risk Assessment
Preparation and Handling of Reagent - Can be prepared on the open bench. There is no apparent hazard associated with this reagent
Spillage - Mop up with paper towels and place in yellow bag .
Disposal - The reagent can be flushed down the sink with excess water .
i. Stock Acrylamide Bis Solution:
Acrylamide 30g Bisacrylamide 0.8g Dowex AG1 Resin 1g Water to 100 ml Store in dark bottle at 4'C. Can be used up to one month. Prior to use filter off Resin.
Risk Assessment
Preparation of Reagent - Both Acrylamide and Bisacrylamide are neurotoxic and can be absorbed through the skin. Wear gloves and a face mask when weighing out these two compounds. Weigh out in a fume cupboard. Wash hand prior to removing gloves. Wash any exposed skin that comes in contact with these compounds IMMEDIATELY.
Handling of the Reagent - The solution is as toxic as the compounds. Wear gloves at all times when handling the reagent. Wash hand prior to removing gloves. Wash any exposed skin that comes in contact with these compounds IMMEDIATELY.
Spillage - Mop up any spillage with paper towels which are then double bagged. Wash spillage area well with water and detergent.
Disposal - Pour contents carefully into a beaker. Add 5ml of 1.5% Ammonium Persulphate solution and 50 ul of TEMED per 20ml of reagent Cover beaker and allow to polymerize over night. Dispose of gel in double yellow bag. There maybe so free acrylamide still present so handle gel wearing gloves.
ii: Ammonium Persulphate Solution: 1.5%
Ammonium Persulphate 150mg Water to 10 ml Make up fresh prior to use.
Risk Assessment
Preparation of Reagent - Ammonium Persulphate is an 'irritant'. Weigh out in fume cupboard.
Handling of Reagent - Little apparent hazard associated with this reagent.
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
iii: Sodium Dodecyl Sulphate (SDS) Solution: 10%
Sodium Dodecyl Sulphate 1g Water to 10 ml Stable at room temperature, precipitates out at 4'C
Risk Assessment
Preparation of Reagent - Sodium Dodecyl Sulphate generates irritant dust when being weighed out. Weigh out in fume cupboard wearing face mask. Add it carefully to the other components (preferably after addition of water).
Handling of Reagent - Little apparent hazard associated with this reagent.
Spillage - Mop up with paper towel, large spillage with mop & bucket
Disposal - Flush down sink with excess water.
i: Stacking Gel (20ml)
Acrylamide Bis Solution 2.5 ml Stacking Gel Buffer 5.0 ml 10% SDS 0.2 ml 1.5% Ammonium Persulphate 1.0 ml TEMED 15 µl Water 11.3 ml De-aerate under vacuum prior to addition of Ammonium Persulphate or TEMED.
NOTE: WEAR GLOVES THROUGH OUT PREPARATION PROCEDURE
Risk Assessment
Preparation of Reagent - Due to the presence of Acrylamide Bis solution, wear gloves through out the preparation of this reagent. This reagent can be prepared and used on the open bench.
Handling of the Reagent - Wear gloves when handling this reagent. When pouring avoid generation of aerosols.
Spillage - Mop up with paper towels which are disposed of in the yellow bag. Clean contaminated area well with water and detergent.
Disposal - Allow reagent to gel and dispose of solid in yellow bag. Because there maybe unpolymerised acrylamide present in the gel, always handle wearing gloves.
ii: Resolving Gel (30 ml)
Composition depends on final Acrylamide Concentration.
Volume of Reagents in ml unless stated otherwise
|
Reagents |
20.0% |
17.5% |
15.0% |
12.5% |
10.0% |
7.5% |
5.0% |
|
Acrylamide/Bis Solution |
20 |
17.5 |
15 |
12.5 |
10 |
7.5 |
5 |
|
Resolving Buffer |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
|
10% SDS |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
0.3 |
|
1.5% Ammonium Persulphate |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
|
Water |
4.45 |
6.95 |
9.45 |
11.95 |
14.45 |
16.95 |
19.45 |
|
TEMED |
15µl |
15µl |
15 µl |
15 µl |
15µl |
15 µl |
15 µl |
Gel are de-aerated prior to addition of Ammonium Persulphate and TEMED.
NOTE: WEAR GLOVES THROUGH OUT PREPARATION PROCEDURE.
Risk Assessment
Preparation of Reagent - Due to the presence of Acrylamide Bis solution, wear gloves through out the preparation of this reagent. This reagent can be prepared and used on the open bench.
Handling of the Reagent - Wear gloves when handling this reagent. When pouring avoid generation of aerosols.
Spillage - Mop up with paper towels which are disposed of in the yellow bag. Clean contaminated area well with water and detergent.
Disposal - Allow reagent to gel and dispose of solid in yellow bag. Because there maybe unpolymerised acrylamide present in the gel, always handle wearing gloves.
i: Stacking Gel (20ml)
Acrylamide Bis Solution 2.5 ml Stacking Gel Buffer 5.0 ml 1.5% Ammonium Persulphate 1.0 ml TEMED 15µl Water 11.5 ml De-aerate under vacuum prior to addition of Ammonium Persulphate or TEMED.
NOTE: WEAR GLOVES THROUGH OUT PREPARATION PROCEDURE.
Risk Assessment
Preparation of Reagent - Due to the presence of Acrylamide Bis solution, wear gloves through out the preparation of this reagent. This reagent can be prepared and used on the open bench.
Handling of the Reagent - Wear gloves when handling this reagent. When pouring avoid generation of aerosols.
Spillage - Mop up with paper towels which are disposed of in the yellow bag. Clean contaminated area well with water and detergent.
Disposal - Allow reagent to gel and dispose of solid in yellow bag. Because there maybe unpolymerised acrylamide present in the gel, always handle wearing gloves.
ii. Resolving Gel (30 ml)
Composition depends on final Acrylamide Concentration.
Volume of Reagents in ml unless stated otherwise
|
Reagents |
20.0% |
17.5% |
15.0% |
12.5% |
10.0% |
7.5% |
5.0% |
|
Acrylamide/Bis Solution |
20 |
17.5 |
15 |
12.5 |
10 |
7.5 |
5 |
|
Resolving Buffer |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
3.75 |
|
TEMED |
15µl |
15µl |
15µl |
15µl |
15µl |
15µl |
15µl |
|
1.5% Ammonium Persulphate |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
1.5 |
|
Water |
4.75 |
7.25 |
9.75 |
12.25 |
14.75 |
17.25 |
19.75 |
Gel are de-aerated prior to addition of Ammonium Persulphate and TEMED.
NOTE: WEAR GLOVES THROUGH OUT PREPARATION PROCEDURE.
Risk Assessment
Preparation of Reagent - Due to the presence of Acrylamide Bis solution, wear gloves through out the preparation of this reagent. This reagent can be prepared and used on the open bench.
Handling of the Reagent - Wear gloves when handling this reagent. When pouring avoid generation of aerosols.
Spillage - Mop up with paper towels which are disposed of in the yellow bag. Clean contaminated area well with water and detergent.
Disposal - Allow reagent to gel and dispose of solid in yellow bag. Because there maybe unpolymerised acrylamide present in the gel, always handle wearing gloves.
Set up Bio Rad Electrophoresis cassette with 18 * 16 cm plates and 1.5 mm or 0.75 mm spacers. Prepare resolving gel using composition in appropriate table; only half the solution volumes are needed for the cassettes with 0.75mm spacers. Using 10 or 20 ml syringe, inject about 25 ml of resolving gel solution between the plates. Overlay with a few ml of water and allow to polymerize. This should not take longer than 30 minutes. If it does, the reagents have probably gone off and preparation should be repeated with fresh reagents.
Tip off water overlay and insert spacer comb into top of cassette. Prepare stacking gel solution and using 10 ml syringe inject to top of cassette. Allow to gel for one hour. The gel can now be used but better results maybe obtained if the gel is allowed to 'age' over night at 4'C.
Risk Assessment of Method
Wear gloves at all handling stages of this method. Wash hands prior to removal of gloves. Dispose of unused or unwanted polymerised gel in yellow bag.
i: Cracking Solution for general Protein Analysis :
Sodium Dodecyl Sulphate 1g Mercaptoethanol 2 ml Bromophenol Blue 10mg Stacking Buffer stock 1.5 ml Sucrose 5g Water 6.5 ml (For analysis where tertiary structure is important, omit the mercaptoethanol)
Risk Assessment
Preparation of Reagent - Weigh out sodium dodecyl sulphate in the fume cupboard. Due the unavoidable generation of irritant dust, wear a face mask. Add mercaptoethanol to other reagents also in the fume cupboard. Store reagent in UC with tight fitting cap.
Handling of Reagent - Open and dispense from container in fume cupboard. There should never be a smell of mercaptoethanol in the laboratory. Wear gloves when handling this reagent
Spillage - Mop up with paper towel which is disposed of in a double yellow bag.
Disposal - Place container in yellow sharps box of incineration. Do not pour contents down the sink.
Method :
Mix 1 part of cracking solution with 4 part of sample in 1.5cm Eppendorf centrifuge tube, cap and pierce cap with needle hole. Place in a rack which in turn is place in a boiling water bath for about 5 minutes. Cool and where necessary (ie with whole bacteria) vortex and spin at 13,000rpm for 10 minutes to remove insoluble debris. Apply in 5-40 ml aliquot to gel slot.
Risk Assessment of Method
Add cracking solution to sample in fume cupboard. Avoid exposure to mercaptoethanol fumes. When no longer needed, 'cracked' solutions are disposed of into the yellow bag.
i: High Density Sucrose Solution :
Sucrose 5g Stacking Buffer stock 1.5ml Bromophenol Blue 10 mg Water 8.5 ml Add one part of reagent to 4 parts of sample
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water
Remove sample comb from top of gel cassette and insert cassette into upper reservoir. Put about 4l of single strength reservoir buffer in lower reservoir, fit upper reservoir (with gel cassettes attached) and place 1l of single strength reservoir buffer in upper reservoir. Using fine pipette tip and automatic pipette apply between 5-30µ l of sample. Because of the sucrose the sample will sink to the upper gel surface. Close system and fit electrodes to transformer. Set voltage to about 100v. After half an hour, once sample has completely entered stacking gel, increase voltage to 250v. The run is complete when the bromophenol blue marker is about 5mm from the bottom of the gel. (Unless of course one is running high molecular weight proteins when it maybe necessary to prolong the separation time). Remove the cassette from upper reservoir and pour electrophoresis buffer down the sink. Avoid opening the cassette with the aid of any metal implement such a metal spatula. Use a plastic spatula or the flat end of a disposable scalpel handle. Remove gel from plate and proceed to next step which involves detection of separated material either by direct staining or after blotting.
Risk Assessment of Method
There is little hazard associated with the above procedure. Gloves should be worn when handling the exposed gel due to the possibility of the presence of unpolymerised acrylamide. Sensitive staining methods involving silver will detect fingerprints.
(Schägger and Jagow 1987)
By changing the buffer system improved resolution down to 1kDa can be achieved
A: Buffer Systems
i: Anode Buffer: 200mM Tris HCl pH 8.9
Tris 96.8g HCl 185 ml Water to 4l Check pH and adjust accordingly
Risk Assessment
Preparation of Reagent - Can be prepared on the open bench. 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - There is no apparent hazard associated with this reagent
Spillage - Mop up with paper towels and place in yellow bag .
Disposal - The reagent can be flushed down the sink with excess water .
ii: Cathode Buffer 100mM Tris/100m Tricine with 0.1% SDS pH 8.25
Tris 12.1g Tricine 17.9g SDS 1g Water to 1l
Risk Assessment
Preparation of Reagent - Sodium Dodecyl Sulphate generates irritant dust when being weighed out. Weigh out in fume cupboard wearing face mask. Add it carefully to the other components (preferably after addition of water).
Handling of Reagent - Little apparent hazard associated with this reagent.
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
iii: Gel Buffer. 3M Tris/HCl with 0.3% SDS pH 8.45
Tris 36.3g HCl (2M) 50ml Sodium Dodecyl Sulphate (SDS) 300mg Water to 100ml Check pH and adjust accordingly
Risk Assessment
Preparation of Reagent - Sodium Dodecyl Sulphate generates irritant dust when being weighed out. Weigh out in fume cupboard wearing face mask. Add it carefully to the other components (preferably after addition of water). 2M HCl is corrosive and should be handled wearing gloves.
Handling of Reagent - Little apparent hazard associated with this reagent.
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
i. Stock Acrylamide Bis Solution with 2.7% Cross linker:
See standard gel procedure for composition, preparation and handling
ii. Stock Acrylamide Bis Solution with 6% Cross linker:
Acrylamide 28.2g Bisacrylamide 1.8g Dowex AG1 Resin 1g Water to 100 ml Store in dark bottle at 4'C. Can be used up to one month. Prior to use filter off Resin.
Risk Assessment
Preparation of Reagent - Both Acrylamide and Bisacrylamide are neurotoxic and can be absorbed through the skin. Wear gloves and a face mask when weighing out these two compounds. Weigh out in a fume cupboard. Wash hand prior to removing gloves. Wash any exposed skin that comes in contact with these compounds IMMEDIATELY.
Handling of the Reagent - The solution is as toxic as the compounds. Wear gloves at all times when handling the reagent. Wash hand prior to removing gloves. Wash any exposed skin that comes in contact with these compounds IMMEDIATELY.
Spillage - Mop up any spillage with paper towels which are then double bagged. Wash spillage area well with water and detergent.
Disposal - Pour contents carefully into a beaker. Add 5ml of 1.5% Ammonium Persulphate solution and 50µl of TEMED per 20ml of reagent Cover beaker and allow to polymerize over night. Dispose of gel in double yellow bag. There maybe so free acrylamide still present so handle gel wearing gloves.
|
Stacking gel 4% T, 3%C |
Spacer gel 10%T,3%C |
10%T, 3%C |
16.5%T 3% C |
16.5%T 6%C |
16.5%T 6% C 6M Urea | |
|
Stock with 3% C |
2.5ml |
5ml |
10ml |
16.5ml |
- |
- |
|
Stock with 6% C |
- |
- |
- |
- |
16.5ml |
16.5ml |
|
Gel Buffer |
5ml |
5ml |
10ml |
10ml |
10ml |
10ml |
|
Glycerol |
- |
- |
4g |
4g |
4g |
- |
|
Urea |
- |
- |
- |
- |
- |
10.8g |
|
1.5% Ammonium Persulphate |
1ml |
0.75ml |
1.5ml |
1.5ml |
1.5 |
- |
|
TEMED |
15µl |
7.5µl |
15µl |
15µl |
15µl |
15µl |
|
Water |
11.5 |
4.25ml |
to 30 ml |
to 30 ml |
to 30 ml |
to 30 ml |
|
Total Volume |
20ml |
15ml |
30ml |
30ml |
30ml |
30ml |
Risk Assessment
Preparation of Reagent - Due to the presence of Acrylamide Bis solution, wear gloves through out the preparation of this reagent. This reagent can be prepared and used on the open bench.
Handling of the Reagent - Wear gloves when handling this reagent. When pouring avoid generation of aerosols.
Spillage - Mop up with paper towels which are disposed of in the yellow bag. Clean contaminated area well with water and detergent.
Disposal - Allow reagent to gel and dispose of solid in yellow bag. Because there maybe unpolymerised acrylamide present in the gel, always handle wearing gloves.
This are volumes for 1.5mm spacer 16*18cm Cassette - For other sizes adjust accordingly
ii: Preparation of 10%T, 3%C Gel - General purpose gel can be used instead of 12.5% gel with Laemmli system.
Set up Bio Rad Electrophoresis cassette with 18 * 16 cm plates and 1.5 mm or 0.75 mm spacers. Using 10 or 20 ml syringe, inject about 30 ml of 10%T, 3%C separating gel solution between the plates. Overlay with a few ml of water and allow to polymerize. This should not take longer than 30 minutes. If it does, the reagents have probably gone off and preparation should be repeated with fresh reagents.
Tip off water overlay and insert spacer comb into top of cassette. Prepare stacking gel solution and using 10 ml syringe inject to top of cassette. Allow to gel for one hour. The gel can now be used but better results maybe obtained if the gel is allowed to 'age' over night at 4'C.
iii: Preparation of 16.5% gels - These are used to separate lower molecular weight proteins and peptides.
Molecular Range 16.5%, 3%T - 2-68 kDa
16.5%,6%T -1.5- 45 kDa
16.5%,6%T -1.5-35 kDa.
Set up Bio Rad Electrophoresis cassette with 18 * 16 cm plates and 1.5 mm or 0.75 mm spacers. Mark of on plate 11cm from bottom (mark 1) and 13cm From bottom (mark 2) and set plates in holder. Add 16.5% gel (either 3%C, 6%C or 6%C+urea) separating solution to mark 1. Carefully overlay with 10% stacking gel to mark 2 and overlay this with water and allow both gels to polymerise.
Tip off water overlay and insert spacer comb into top of cassette. Prepare stacking gel solution and using 10 ml syringe inject to top of cassette. Allow to gel for one hour. The gel can now be used but better results maybe obtained if the gel is allowed to 'age' over night at 4'C.
Risk Assessment of Method
Wear gloves at all handling stages of this method. Wash hands prior to removal of gloves. Dispose of unused or unwanted polymerised gel in yellow bag.
Methodology is similar to that with the Laemmli system except a separate Anode and Cathode buffer system is used. Start with 100v constant for 30min-1hour and then increase voltage to about 250v. The original method used lower voltage settings (30v to 100v) but separation times take very long and resolution does not appear to be improved.
Risk Assessment of Method
There is little hazard associated with the above procedure. Gloves should be worn when handling the exposed gel due to the possibility of the presence of unpolymerized acrylamide. Sensitive staining methods involving silver will detect fingerprints.
Hames BD (1990) One dimensional polyacrylamide gel electrophoresis in Gel
Electrophoresis of Proteins: A practical approach Ed BD Hames and D Rickwood Pub
IRL Press, Oxford 2rd Edition pp 1-147 (particular reference pp 30 - 50)
This reference was used for buffer and gel preparation receipts.
Schägger H and von Jagow G (1987) Tricine-Sodium Dodecyl Sulphate -
Polyacrylamide Gel Electrophoresis for the separation of Proteins in the range
from 1 - 100kDa
Analytical Biochemistry 166: 368-379
The following hazardous chemicals are used in the procedures below :-
Acetic Acid (glacial) - Corrosive.
Copper Chloride - Harmful
Ethanol - Inflammable
Formaldehyde - Toxic
Methanol - Toxic
Silver Nitrate - Toxic
Trichloroacetic Acid - Corrosive
Zinc Chloride - Corrosive
i: Reagents :
a: Coomassie Blue Stain:
Coomassie Blue 600 mg Water 260 ml Methanol 240 ml Acetic Acid 100 ml Make up at least 1 hour prior to use. Filter through Whatman's No 1 filter paper.
Risk Assessment
Preparation of reagent : Wear gloves while preparing this reagent. Both Acetic Acid and Methanol give off either irritant or harmful fumes and potential exposure should be kept to a minimum (less than 5 minutes). Coomassie blue can be weighed out on the open bench but wear gloves and avoid inhaling dust.
Prepared reagent : This reagent is irritant and can give off harmful fumes. Always store in a closed container which is well marked as 'irritant'.
Spillage: Mop up with paper towels and dispose of towels in yellow bags. Large spillages can be disposed down the sink with excess water after neutralization of acid.
Disposal: Dilute with excess water and neutralize pH to above 7 with 1M sodium hydroxide. Flush down sink with further excess water.
b: De-stain Solution:
Water 1200 ml Methanol 800 ml Acetic Acid 200 ml Note it has been found that sometimes, 1:1 water methanol (which is usually recommended) causes shrinkage of gel and leaching of stain. Therefore 1.2:0.8 ratio is recommended
Risk Assessment - Similar to staining solution without reference to dye
ii: Method
Place gel in stain for at least 4 hours or if more convenient, over night. Discard staining solution down sink with plenty of water. Transfer to de-staining reagent and change at hourly intervals until background is clear.
An alternative method of de-staining has been proposed by G Hervieu (Elsevier Trends Journal Technical OnLine, http://tto.trends.com/Tip TO1089.- Electrophoresis section. Suspend the stained gel in 500ml of distilled water in a large glass dish with loose fitting lid (a large casserole dish can be used). Place in microwave oven and heat at full power for between 15-25 minutes. Remove water (which will be very hot) with aid of water pump. It has been suggested that the method may not work well with protein below the size of 20-30Kd.
Store in de-stain, 7% acetic Acid or dry gel (see later)
Risk Assessment
Staining and de-staining is carried out in containers with tight fitting lid in a well ventilated area of the laboratory. Wear gloves when ever handling gel or stain. The dried gels can be handled without protection.
iii: Reference
Hames BD (1990) One dimensional polyacrylamide gel electrophoresis in Gel Electrophoresis of Proteins: A practical approach Ed BD Hames and D Rickwood Pub IRL Press, Oxford 2rd Edition pp 1-147 ( reference for above stain is on page 53)
i: Reagents :
a: Coomassie Blue Stain:
Coomassie Blue 40 mg Water 230 ml Methanol 220 ml Acetic Acid 50 ml Make up at least 1 hour prior to use. It is important than excess methanol is not used
Risk Assessment
Preparation of reagent : Wear gloves while preparing this reagent. Both Acetic Acid and Methanol give off either irritant or harmful fumes and potential exposure should be kept to a minimum (less than 5 minutes). Coomassie blue can be weighed out on the open bench but wear gloves and avoid inhaling dust.
Prepared reagent : This reagent is irritant and can give off harmful fumes. Always store in a closed container which is well marked a 'irritant'
Spillage: Mop up with paper towels and dispose of towels in yellow bags. Large spillages can be disposed down the sink with excess water after neutralization of acid
Disposal: Dilute with excess water and neutralize pH to above 7 with 1M sodium hydroxide. Flush down sink with further excess water.
ii: Method
Place gel in stain for over night. The gel can be stored in the same solution until it is to be dried
Risk Assessment
Staining and de-staining is carried out in containers with tight fitting lid in a well ventilated area of the laboratory. Wear gloves when ever handling gel or stain. The dried gels can be handled without protection.
iii: Method Reference
H Chen, H Cheng and M Bjerknes (1993) One step Coomassie Brilliant Blue R250 Staining of protein in polyacrylamide gel 212 295- 296
This method was developed in house when it was found that the Commassie blue reagent used to assay proteins (often used to assay protein in samples prior to their electrophoresis) could be used to rapidly stain proteins in gels following native or SDS gel electrophoresis. Improved results were obtained following washing in a fixing reagent to remove SDS and washing the stained gels in water overnight. The reagent is based on that of Read and Northcote (1981)
Stock Dye Solution
Coomassie Blue G 330 mg Phosphoric Acid/Ethanol (2:1 v/v) 100ml Dissolve the dye in the phosphoric acid/ethanol mixture and store in a brown bottle at room temperature.
Risk Assessment
Preparation and handling of reagent : Phosphoric acid is corrosive. The reagent can be prepared on the open bench but wear gloves when preparing or handling the reagent.
Spillage: Mop up with paper towel and dispose in yellow bag for incineration. Wash area of spillage with soapy water.
Disposal: Dilute the reagent 50 fold and neutralise with sodium Hydroxide. Flush neutralised solution down sink with excess water.
Working Dye solution
Stock Dye Reagent 4 ml Phosphoric Acid 8 ml Ethanol 3.75 ml Water to 100 ml
Make up working solution on day of use
Risk Assessment
Preparation and handling of reagent : Phosphoric acid is corrosive. The reagent can be prepared on the open bench but wear gloves when preparing or handling the reagent.
Spillage: Mop up with paper towel and dispose in yellow bag for incineration. Wash area of spillage with soapy water.
Disposal: Dilute the reagent 50 fold and neutralise with sodium Hydroxide. Flush neutralised solution down sink with excess water.
Fixing Solution: Fixing Reagent: (40% Ethanol, 10% Acetic Acid)
Ethanol 200 ml Acetic Acid 50 ml Water - 250 ml
Risk Assessment
Preparation and handling of reagent : Wear gloves while preparing this reagent. Acetic Acid gives off either irritant or harmful fumes and potential exposure should be kept to a minimum (less than 5 minutes.)
Prepared reagent:This reagent is irritant and can give off harmful fumes. Always store in a closed container which is well marked as 'irritant.
Spillage: Mop up with paper towels and dispose of towels in yellow bags. Large spillage can be disposed down the sink with excess water after neutralisation of acid.
Disposal: Dilute with excess water and neutralise pH to above 7 with 1M sodium hydroxide. Flush down sink with further excess water.
Risk Assessment
Staining and de-staining is carried out in containers with tight fitting lid in a well ventilated area of the laboratory. Wear gloves when ever handling gel or stain. The dried gels can be handled without protection.
Reference
Read SM and Northcote DH (1981) Minimization of variation in response to different proteins of the Coomassie Blue G dye-binding assay for protein. Analytical Biochemistry 116: 53-64
The following hazardous reagents were used in this procedure.
Acetic Acid Corrosive
Formaldehyde Toxic
Glutaraldehyde Toxic
Silver Nitrate Toxic
i: Fixing Reagent: (40% Ethanol, 10% Acetic Acid)
Ethanol 200 ml Acetic Acid 50 ml Water - 250 ml
Risk Assessment
Preparation of reagent : Wear gloves while preparing this reagent. Acetic Acid gives off either irritant or harmful fumes and potential exposure should be kept to a minimum (less than 5 minutes.
Prepared reagent : This reagent is irritant and can give off harmful fumes. Always store in a closed container which is well marked as 'irritant.
Spillage: Mop up with paper towels and dispose of towels in yellow bags. Large spillages can be disposed down the sink with excess water after neutralisation of acid.
Disposal: Dilute with excess water and neutralise pH to above 7 with 1M sodium hydroxide. Flush down sink with further excess water.
ii: Washing Reagent:
Ethanol 400 ml Water 600 ml
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
iii: Fix/Sensitising Solution
Glutaraldehyde (25%) 200µl Formaldehyde (37%) 10µl Ethanol 40ml Water to 100ml
Risk Assessment
Preparation of reagent : Wear gloves while preparing this reagent and make up in the fume cupboard.
Prepared reagent : The final concentrations of both glutaraldehyde and formalin mean that the reagent can be handled on the open bench. However use in closed container when staining
Spillage: Mop up with paper towels and dispose of towels in yellow bags. Large spillages can be disposed down the sink with excess water.
Disposal: Dilute with excess water and flush down sink with further excess water.
iv: Sensitising Reagent
Sodium thiosulphate 40mg Water to 200ml
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
v: Stock Silver Nitrate Solution: (20%)
Silver Nitrate 2 g Water (distilled) - 10 ml Make up and store in dark glass bottle. Dilute to 0.1% (200 fold for use in staining.)
Risk Assessment
Preparation of Reagent - Silver Nitrate is corrosive and toxic. Weigh out in fume cupboard. Wear gloves.
Handling of Reagent - Wear gloves when ever handling this reagent.
Spillage - Mop up with paper towels which are disposed of in yellow bag.
Disposal - Dilute 1000 fold prior to flushing down sink with excess water.
vi: Developer Solution
Sodium Carbonate 5.0 g Formaldehyde 40µl Water 200 ml Make up just before use.
vii: Risk Assessment
Preparation of Reagent Formaldehyde vapour is toxic and corrosive Add formaldehyde to solution in the fume cupboard. Wear gloves during preparation of reagent.
Handling of reagent Amount of formaldehyde present in the reagent is so small that the reagent has little or no associated hazard.
Spillage - Mop up onto paper towels which are disposed of in the yellow bags.
Disposal - Flush down sink with excess water
WEAR GLOVES THROUGHOUT THIS PROCEDURE. Use glassware where possible.
1) Place in 200ml of 40% Ethanol /10% Acetic Acid fixing reagent for 10 minutes.
2) Rinse in 200ml of distilled water for 10 minutes
3) Place in 100ml of Fix/sensitising reagent for 5 minutes
4) Rinse in 200ml of 40% Ethanol wash reagent for 20 minutes
5) Rinse in 200ml of distilled water for 20 minutes
6) Place in 100ml of sensitising reagent (0.2% thiosulphate) for 1 minute
7) Do two rinses in 200ml of distilled water of 1 minute each
8) Place in 100ml 0.1% Silver nitrate solution for 20 minutes.
9) Rinse 200ml of distilled water for 1 minute.
10) Place in 100ml of developing solution until it turns yellow (approx... 1 minute). Pour off immediately and replace with 100ml fresh developing reagent. Develop until required degree of staining is obtained (approx. 15 minutes).
11) Stop reaction in 5% Acetic acid for 5 minutes. Wet gels are stored in 0.03% sodium carbonate.
Risk Assessment of Method
Staining procedure should be carried out in a well ventilated part of the laboratory. When not in use, staining containers should be tightly closed. Wear gloves at all stages of this procedure.
Swain M and Ross NW. A silver stain protocol for protein yielding high resolution and transparent background in sodium dodecyl sulphate polyacrylamide gels. Electrophoresis 1995; 16: 948-951
Copper Chloride (0.3M)
or
Zinc Chloride (0.3M)Zinc is the more sensitive component but must be used fresh
Risk Assessment
Preparation and Handling of Reagents - Reagents maybe weighed out on the open bench but wear gloves when preparing or handling the reagents.
Spillage - Mop up onto paper towels which are disposed of in the yellow bag.
Disposal - Dilute reagent to content of 5mg/l of metal (2mM for the salt) and flush diluted material down the sink.
Soak Gel with rocking in the 0.3M solution for up to 5 minutes. Background goes turbid, protein bands are clear. View on dark plate with overhead light. Very rapid staining method which stains the background rather than the protein. The pattern will not be preserved on drying but the gels can be counter stained by Coomassie blue staining. The method is restricted to SDS-PAGE.
Risk Assessment of Method
Wear gloves when handling the reagent or the 'stained' gel. Dispose of gel in yellow bag for incineration.
Dzandu JK, Johnson JF and Wise GE (1988) Sodium Dodecyl Sulfate Gel Electrophoresis: Staining of polypeptides using heavy metal salts. Analytical Biochemistry 174 157 -167.
An alternative method has been described which because there is a pre-soak stage with SDS buffer, may be used with non-SDS gels as well.
200mM Imidazole containing 0.1% SDS
Imidazole 2.72 g Sodium Dodecyl Sulphate 0.2g Water to 200ml
Risk Assessment
Preparation of Reagent - Sodium Dodecyl Sulphate generates irritant dust when being weighed out. Weigh out in fume cupboard wearing face mask. Add it carefully to the other components (preferably after addition of water).
Handling of Reagent - Little apparent hazard associated with this reagent
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
200mM Zinc Sulphate
Zinc Sulphate 5.74g Water to 200ml
Risk Assessment
Preparation and Handling of Reagent - Little apparent hazard associated with the preparation or handling of reagent
Spillage - Mop up with paper towel, large spillage with mop & bucket.
Disposal - Flush down sink with excess water.
- Rinse gel for 30-60 seconds in distilled water
- Soak gel in Imidazole/SDS reagent for 10 minutes with continuous shaking.
- Transfer gel to Zinc Sulphate solution and shake until white background fully develops (usually under 1 minute)
- Wash gel in distilled water and photograph using reflective light on a black background
Risk Assessment of Method
Wear gloves when handling the reagent or the 'stained' gel. Dispose of gel in yellow bag for incineration.
Hardy E, Santana H, Sosa A, Henández L, Fernández-Patrón C, Castellanos-Serra L (1996) Imidazole-Sodium Dodecyl Sulphate -Zinc (Reverse Stain) on Sodium dodecyl sulphate gels. Analytical Biochemistry 240:252-5 (based on original procedure described in Fernández-Patrón C et al (1992) BioTechniques 12:564-73)
The following chemicals used in this procedure are hazardous
Acetic Acid Corrosive
Sypro-Orange Harmful
7.5% Acetic Acid
Acetic Acid 75ml Water to 1l
Risk Assessment:
Preparation and Use of Reagent: Wear gloves and protective clothing when preparing the reagent. It may be prepared on the open bench provided there is adequate ventilation otherwise prepare in fume cupboard. The prepared reagent should be handled wearing gloves.
Spillage: Mop up with paper towels and dispose into yellow bag
Disposal: Pour down sink with excess water.
Sypro-Orange Reagent.
Sypro-Orange solution 10µl 7.5% Acetic Acid 50ml
Ensure that reagent solution is thawed out (it is suspended in DMSO which freezes at 4'C). Vortex mixture before use. The diluted reagent is stored in the dark before use. If staining is to be carried out before blotting, substitute the transfer (Towbin) buffer (see blotting protocol for preparation of transfer buffer) for 7.5% Acetic acid. The reagent can be used at least twice if stored in the dark between uses.
Risk Assessment:
Preparation and Use of Reagent: Wear gloves and protective clothing when preparing and handling this reagent.
Spillage: Mop up with paper towels and dispose into yellow bag
Disposal: Pour down sink with excess water.
If SDS was used in the gel, soak gel for 15 minutes in 300ml of 7.5% Acetic acid (or transfer buffer if one is going to do a subsequent blot) with shaking. Transfer gel to plastic container and add diluted Sypro-Orange reagent. Cover container with aluminium foil and allow to stand for 30 minutes. Pour off reagent and store in dark for subsequent use. Wash gel in 200 ml of fresh 7.5% Acetic acid (or transfer buffer) for several minutes and then photograph under uv light.
COMMENT: I have failed to get this method to work well in my laboratory. It maybe that the wavelength of the uv viewer is more critical than expected. We use the same view as is used for viewing Ethidium Bromide stained gels.
Risk Assessment of method:
The sypro-orange dye may be harmful, always wear gloves when handling the reagent and stained gel.
Steinberg TH, Hangland RP, Singer VI (1996) Applications of SYPRO Orange and SYPRO Red Protein Gel Stains Analytical Biochemistry 239: 238-45.
The follow hazardous chemicals are use in the procedure below :-
Dimethylformamide - Harmful
Methanol - Toxic
Nitroblue Tetrazolium Salt - Harmful
Sodium Carbonate - Harmful
Tris - Irritant
Kodak GBX Developer - Harmful
Transblot Buffer (25mM Tris, 192mM Glycine in 20% Methanol pH 8.3(Towbin transfer buffer - PNAS 76 4350 (1979):
Tris 9.09g Glycine 43.2g Methanol 600ml Water to 3l
Risk Assessment
Preparation of Reagent - Wear gloves when preparing this reagent. Avoid excessive exposure to Methanol vapour or aerosols but can be prepared on open bench with little risk.
Handling of Reagent - Wear gloves when ever handling this reagent.
Spillage - Mop up on paper towels and dispose into yellow bag.
Disposal - Flush down sink with excess water after prior diluting reagent.
Transfer of protein to nitrocellulose sheet
WEAR GLOVES THROUGHOUT THIS PROCEDURE - UNPOLYMERISED ACRYLAMIDE MAY BE PRESENT - ALSO FINGERPRINTS MUST BE AVOIDED
Cut nitrocellulose sheets to size of gel to be blotted. Cut two sheets of Whatman 3MM filter paper to size also. Soak nitrocellulose sheet in transfer buffer for 30 minutes. Also soak filter -paper and scotch brite pads prior to use. Wear gloves when handling wet material or gel to avoid contaminating with fingerprint protein. Place sheet of filter paper on gel with one scotch brite pad on top of paper. Avoid entrapment of air bubbles. Careful place nitrocellulose sheet on other surface again avoid entrapment of air bubbles. Place second filter paper sheet and scotch brite pad on the top and insert whole assembly into plastic cassette. Place remaining buffer and cassette into transblot tank. Orientation of cassette is such that nitrocellulose sheet side is towards the anode plane. Transblot with 30v and 1Amp current over night. Remove nitrocellulose strip and cut up according to parts that are to be stained by different techniques.
Risk Assessment of Method
Wear gloves at all handling stages of this procedure. Ensure transformer has been turned off prior to opening transblotter
Towbin transfer buffer pH 8.3
Tris 3.03g Glycine 14.4g Methanol 200ml Water to 1l Chill buffer prior to use. Omit methanol if using nylon membrane
Risk Assessment
This is little or no hazard associated with preparation or use of this buffer
For other buffers see manual (for DNA transfer use 1 in 10 dilution of 5*TBE buffer)
WEAR GLOVES THROUGH OUT THIS PROCEDURE TO AVOID CONTAMINATION OF MEDIA WITH FINGERPRINTS
1) Soak electrophoretic gel in 500ml of buffer for 15 minutes for .75mm thick gel and 30 minutes for 1.5mm thick gel. This step helps remove some of the electrophoretic buffers and detergents and cause shrinkage caused by methanol in the buffer
2) Cut six pieces of 3MM filter paper to size of gel and one piece of membrane and soak all in buffer for up to 30 minutes Check to ensure there are no air bubbles trapped in either paper or membrane.
3) Pile 3 pieces of filter paper on top of each other on bottom. Use pipette or roller to ensure there are no air bubbles trapped between plate and filter or between filter paper layers.
4) Transfer membrane to filter paper pile and again roll (press) out any trapped air bubbles.
5) Carefully place and align electrophoresis gel on to membrane (roll again).
6) Place remaining 3 pieces of filter paper on top of gel.
7) Carefully place cathode plate on top of stack and engage latches to secure.
8) Replace safety cover and connect to power supply.
Risk Assessment
Gloves are worn through out this procedure more to protect the media than the operator. However polyacrylamide gels could contain unpolymerised acrylamide residues and gloves will protect the operator when handling such gels. Otherwise there is little or no hazard associated with this procedure.
1) For large gels use 20v for about one hour (do not exceed a PD of 25V) and/or a current limit of 3mA/cm2 and a current limit should be set on the transform to avoid over heating. Measure gel prior to inserting placing on membrane and estimate current limit.
2) The period of 1 hour is arbitrary and may need to be extended. This can only be established by practice.
3) At end of run, turn off power, remove safety lid and cathode plate. Carefully remove top filter paper. Transfer gel to Coomassie blue staining solution to establish amount of transfer. Remove membrane and treat according to need. Filter papers can be disposed into yellow bags. Clean anode and cathode plates.
Risk Assessment
As current can only flow when safety lid is secure, there should be no hazard with using this equipment. AVOID USING TOO HIGH A CURRENT SETTING AS THIS WILL CAUSE EXCESSIVE HEATING IN THE EQUIPMENT. Wear gloves when handling 'blot sandwich'
The following points were stressed in the manual not only with regards safety but to ensure optimum transfer.
1) Do not change composition of buffers as stated in this protocol or the manual
2) Ensure there is no air bubbles trapped in any of the layers of the 'blot sandwich'. Remove them as the pile is built up, not just at the end.
3) Avoid excessive heating during a run. DO NOT USE A PD GREATER THAN 25V. SET current limit to 3mA/cm2
Blocking Solution:
Sodium Chloride 4.5g Tris 600mg Albumin (Bovine) 15.00g Tween 20 250µl Water 500ml Add Albumin after dissolving Tris and Sodium Chloride in the water.
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
Washing Solution:
Sodium Chloride 4.5g Tween 20 250µl Water 500ml
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
Horse Radish Peroxidase-Streptavidin Complex Solution
Blocking solution 10ml Antibody 10µl (1/1000 is again a reasonable dilution to use as a starting point - the dilution required can be optimised by trying out various dilutions on dot-blots of the protein under test)
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
Also Required
ECL Detection Reagents (Amersham)
ECL Film or Paper (Amersham)
Developer and Fixer (Kodak)
Wearing gloves, remove the blot from the cassette and if necessary, cut the nitrocellulose into tracks or sections with clean scissors. Always mark the bottom right hand corner of each section - it is very easy to become confused as to the orientation of each piece! Discard the used gel into the yellow bag for incineration. Place each strip into a sealable container of suitable size and follow the steps listed below. All stages should be gently rocked and are carried out at room temperature. During the course of this work, a hybridization oven may become available and can be used as an alternative to the rocking table.
This is a general protocol for this procedure. It is usual to optimise first and second antibody dilutions and incubation times for use in Western Blots by first using dot-blots. A very full protocol and trouble shooting guide is provided by Amersham in the detection kit.
1: 2 X 1 hour in blocking solution (or overnight)
2: 1 hours - HRP-Streptavidine conjugate (or overnight).
5. 5 x 5 minute washes with washing solution (25ml)
Carry out all further steps in the darkroom. Have all equipment needed to hand.
4. Just before it is required, mix equal volumes of detection solutions 1 & 2 together, and add to the blot.
5. Incubate for 1 minute at room temperature, then, working quickly, drain off excess detection reagent by holding the blot vertically and touching the edge against tissue paper. Place onto a clean glass plate and cover with clingfilm. Be careful to smooth out any wrinkles and air pockets.
6. Place the glass plate into a film cassette. All the rest of the steps must take place under RED safelight illumination (not the normal Orange Safelight). Cut a piece of Hyperfilm or Hyperpaper ECL to the correct size and place in contact with the wrapped blot. Cover with another glass plate and shut the cassette.
7. Allow to expose for 15 seconds (FILM) or 5 minutes (PAPER), then quickly remove and replace the film, and develop the exposed film or paper. Estimate a reasonable exposure for the second piece of film by reference to this first exposure result.
8: The ECL Film or Paper is then developed in the darkroom continue to use RED safelight
9: Soak film in a 1:5 dilution of KODAK GBX Developer for 1 - 2 minutes or until film has satisfactorily developed.
10: Wash with running water
11: Soak in a 1:5 dilution of Kodak GBX fixer for 3-4 minutes
12: Wash with running water. Film can now be viewed under normal light
Risk Assessment of Method
Wear gloves at all handling stages of this procedure to minimise contact with unpolymerised acrylamide and to avoid contaminating blot with fingerprints. The developer solution is considered harmful and should not come in contact with skin or eyes. All used reagents can be disposed of down the skin with excess water.
Blocking Solution:
Sodium Chloride 4.5g Tris 600mg Albumin (Bovine) 15.00g Tween 20 250µl Water 500ml Add Albumin after dissolving Tris and NaCl in the water.
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
Washing Solution:
Sodium Chloride 4.5g Tween 20 250µl Water 500ml
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
i) Buffer :
Tris 1.21g Sodium Chloride 580mg Magnesium Chloride 100mg Water to 100ml after adjusting pH with 2M HCl.(pH 9.5)
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent.
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
ii) Nitro Blue Tetrazolium Solution (NBT)
Nitroblue Tetrazolium 10mg N,N dimethyl formamide (70%) 1ml
Risk Assessment
Preparing Reagent - Weigh out NBT and dispense formamide in fume cupboard. Wear gloves and keep container closed when not in use.
Handling of Reagent - Wear gloves when handling this reagent.
Spillage - Mop up onto paper towels and dispose into yellow bag.
Disposal - Flush down sink with excess water.
iii) BCIP solution
5-bromo-4-chloro-3-indoyl phosphate 5mg N,N dimethyl formamide (70%) 1.0ml
Risk Assessment
Preparing Reagent - Weigh out BCIP and dispense formamide in fume cupboard. Wear gloves and keep container closed when not in use.
Handling of Reagent - Wear gloves when handling this reagent.
Spillage - Mop up onto paper towels and dispose into yellow bag.
Disposal - Flush down sink with excess water.
iv) Active stain
NBT solution 660ml BCIP solution 330ml Buffer 100ml Active stain solution is used within one hour of preparation.
Risk Assessment
Preparation and Handling Reagent - The reagent can be made up on the open bench but wear gloves when preparing or handling this reagent.
Spillage - Mop up onto paper towels which are then disposed into a yellow bag.
Disposal - Flush down sink with excess water.
i) Buffer : 50mM Acetate pH 7.5
Sodium Acetate 410mg Water to 100ml pH is adjusted to 7.5 with 1M Acetic acid (one or two drops only will be needed)
Risk Assessment
Preparation and Handling of the Reagent - There is no apparent hazard associated with preparing or handling this reagent. Wear gloves while handling the 1M Acetic acid and avoid inhaling fumes
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
ii) Stock Amino-9-ethylcarbazole (AEC) reagent
3-amino-9-ethylcarbazole 50mg Dimethylformamide 5ml Store at room temperature in dark (long term storage at 4'C)
Risk Assessment
Preparation and Handling of the Reagent - 3-amino-9-ethylcarbazole is potentially carcinogenic. Wear gloves and protective clothing when preparing and handling this reagent. Weigh out in fume cupboard
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
iii) Working AEC reagent
Stock AEC reagent 2.5ml Acetate buffer pH 7.5 47.5ml 30% Hydrogen Peroxide 25ml The reagent should be made up within an hour of use
Risk Assessment
Preparation and Handling of the Reagent - 3-amino-9-ethylcarbazole is potentially carcinogenic. Wear gloves and protective clothing when preparing and handling this reagent. Hydrogen Peroxide is corrosive and should be handled in a fume cupboard
Spillage - Mop up on paper towels which are disposed in yellow bag.
Disposal - Flush down the sink with excess water.
1: Place strip in lunch box and add 50 ml of blocking buffer and shake for at least 2 hours or to stand overnight at 4'C.
2: Add 40 - 100 ml of anti-sera depending on its strength (or from previous determinations - a 1:100 dilution is a good starting point) and incubate the strip for 2 hours at room temperature.
3: The strip is washed in 5 changes of washing solution over a 30 minute period.
The following steps depend on the nature of the enzyme conjugate:
a) Alkaline Phosphatase:
4a: Incubated with alkaline phosphatase conjugated anti immunoglobulin solution (1:1000 dilution of conjugate in blocking buffer).
5a: The strip is again rinsed in 5 changes of Washing buffer.
6a: The washed strip is then incubated for 15 minutes in the alkaline phosphatase detection stain.
7a: The developed strip is washed and dried
b) Peroxidase
4b: Incubate with peroxidase conjugate anti immunoglobulin solution (1:1000 dilution of conjugate in blocking buffer.
5b: The strip is again rinsed in 5 changes of Washing buffer.
6b: The washed strip is then incubated in 50ml of peroxidase detection working solution at room temperature until red precipitation bands are seen (about 5-10 minutes)
7b: The stain strip is washed with several changes of water and dried.
Risk Assessment of Method
Wear gloves at all handling stages of this procedure to minimise contact with unpolymerised acrylamide and to avoid contaminating blot with fingerprints. All used reagents can be disposed of down the skin with excess water.
i) 1% Potassium Hydroxide
ii) 0.3% Tween 20 in PBS
iii) Stain Solution : 100 ml of Indian Black Ink in 100 ml of PBS
Soak nitrocellulose sheet in 100 ml of 1% KOH for 5 minutes. Rinse immediately in PBS and then wash over 10 minute period with 3 changes (100 ml) of PBS containing 0.3% tween at 37'C. The sheet is then stained in 100ml of staining solution overnight and finally washed with distilled water to de-stain.
Risk Assessment of Method
Wear gloves at all handling stages of this procedure.
Blake MS, Johnson KH, Russell-Jones GJ and Gotschlich EC (1984) A rapid sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Analytical Biochemistry 136 175 - 179
Sutherland MW and Skerritt JH (1986) Alkalie enhancement of protein staining on nitrocellulose. Electrophoresis 7 401 - 406.
The BCIP/NBT method for Alkaline Phosphatase method was taken with some minor modifications from Protein Blotting- A guide to transfer and detection, 2rd Edition Bio Rad
Reagent
5% Glycerol in Water
Soak the stained gel in the glycerol solution with shaking. Then transfer gel to 3MM Filter paper or porous cellulose sheet. Soak sheet or paper in water prior to transfer. Ensure that no air is trapped between gel and sheet. Transfer whole to drier. Dry under vacuum and heating for about 1 to 3 hours.
Risk Assessment - Little or no hazard associated with this method.
Glycerol 8ml Methanol 90ml Water 102ml
Soak stained gel in glycerol/methanol reagent for two hours with gentle shaking. Soak a cellophane sheet (20* 20cm - Chemlab) in water and place on top of a 18*14cm glass plate. Ensure there is no air trapped between the sheet and plate. Transfer gel to the sheet and again ensure that there is no air trapped between the sheet and the gel. Cover the gel with a second cellophane sheet that has been soaked in water an then clamp around all sides of the gel and plate with bulldog clips. Leave to dry out over two days. Remove the bulldog clips. Remove the gel cellophane sandwich from the glass plate. Trim off the excess cellophane.
Risk Assessment - Little or no hazard associated with this method.
The general procedures outline above are used in the following applications.
Organisms Typed
Clostridium Difficle
Burkholderia Cepacia
Shigella Sonni
Klebsiella pneumonia
Stenotrophomonas Maltophilia
Bacteria for typing are best prepared from cultures on plates. Take two loops of culture and suspend in 2 ml of sterile PBS. Mix on vortex mixer until pellet is completely suspended. Take 100 ml of this suspension and add with mixing to 900 ml of PBS. Read at 450 nm against a PBS blank. OD of original suspension is x10 the OD of the reading obtained. Take 1.5 ml of the original suspension and place in Eppendorf centrifuge tube. Spin at 13,000 rpm for 10 minutes. Re-suspend pellet in 1 ml of PBS and re-spin. Further treatment depends on nature of protein to be investigated. Estimate the protein content using the Coomassie Blue method.
a) Whole Cell Protein: The pellet is then taken up in diluted stacking buffer (1 in 4 dilution of stock); the volume of water depending on the original protein content of the specimen. Make up a solution containing 1-2mg/ml. A higher concentration maybe be used for other bacteria but the above is the maximum for P. cepacia before gross interference by nucleic acid.
b) Cell Wall Protein extracted by EDTA. Cell pellet is suspended in 10mM EDTA pH 7.0 and incubated at 45'C for 30 minutes. Cool suspension, spin at 13,000 rpm for 10 minutes. Remove supernatant with care. This method works for C. Difficle but may work for other organisms. (It does not work well for B.cepacia)
To either of the preparations, add cracking solution (1 part to 4 parts supernatant). Heat in boiling water bath for 4 minutes cool, vortex mixture and spin at 13,000rpm for 10 minutes.
Risk Assessment of Method
Consult appropriate risk assessments for Category 1 organisms and the procedures of PAGE. 'Cracking' of the organism will kill the organism and can thence be handle as non-hazardous.
12.5% Acrylamide Gels using the Laemmli system or 10%T, 3%C with the Tricine-SDS buffer systems appear to give the best results. 10 -20 ml of samples are separated, the outer wells are not used. It is carried out until the bromophenol markers are about 1 cm from the bottom. Low weight molecular markers are included on the gel. The Gels are stain with the Coomassie Blue stain.
Risk Assessment - See appropriate Risk Assessments for the steps above.