Derek Wong

Biology 275L-10

Lab 1

January 29, 2002

Analysis of Bacterial Lysates by Electrophoresis

Introduction

When given a sample of bacterial lysates, there are several ways to determine the protein composition. The primary way is to use electrophoresis to separate proteins by molecular weight. Electrophoresis works by running a current though a sample and having it separate into bands along the gel. The theory behind this is that charged molecules will move when placed in an electric field from one side to the other.

In this experiment, the specific method used with be Sodium-Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS-PAGE is a method in which the smaller proteins will move faster than the larger proteins, hence creating a gradient of the heavier proteins towards the center and the lighter proteins toward the end of the gel. The SDS is an anionic detergent which unfold proteins into a random-rod like chain. The negative charge of the SDS is much stronger than the negative and positive charges of the amino acid and therefore the SDS denatured proteins have a net negative charge causing them to migrate to the anode during electrophoresis. The polyacrylamide gel is an innert matrix formed by the cross-linkage of acrylamide. Ammonium persulfate (APS) is used to initate the reaction by the generation of free radicals and tetramedthylethylenediamine (TEMED) is used to accelerate the polymerization of acrylamide.

Once the electrophoresis process is completed, bands will appear representing a particular protein of a specific weight. When ran with a molecular weight standard, one can determine how many proteins make up that one sample, and how much each component way. However, one can not determine what the identity is of each protein because different proteins of different structures may have the same molecular weight. Another application of electrophoresis is that the bands formed can be used to identify the sample. For example, if sample A has a known banning pattern, then if sample B has the exact same banning pattern then it is most likely that sample A and sample B are the same sample.

To determine what the molecular weight of each band, a standard curve is made. The retardation factor (Rf) is found by: Rf = (distance migrated by the molecule)÷(distance migrated by the dye front). A standard curve is then constructed by plotting the known molecular weight versus the Rf value found, and drawing a best-fit line. The best-fit line is used to then determine the relative molecular weights of the unknowns.

The purpose of this experiment is use the SDS-PAGE to compare the various protein profiles of the bacterial lysates and determine the two unknowns, X and Y.

Results

Rf = Distance migrated by the molecule/Distance migrated by the dye front

Table 1: Retardation Factor (Rf) values for the Molecular Weight Standards

Standard	Molecular Weight	log₁₀Molecular Weight	Rf Value
Myosin	224,000 Daltons	5.35	1
Beta-galactosidase	122,000 Daltons	5.08	0.88
Ovalbumin	51,500 Daltons	4.712	0.75
Bovine Serum Albumin	96,000 Daltons	4.982	0.83
Carbonic Annhydrase	35,000 Daltons	4.55	0.58
Lysozyme	21,000 Daltons	4.32	0.12
Soybean Trypsin Inhibitor	28,700 Daltons	4.46	0.30

Table 1: Shows the Retardation Factor (Rf) values, the log₁₀ molecular weights, and the given molecular weights found in the standard.

Table 2: Bacterial Cultures in each well.

Well	Species
A1	E. Coli, lysozyme added
A2	E. Coli
B	S. marcescens, lysozyme added
C1	M. luteus, lysozyme added
C2	M. luteus
D1	B. subtilis, lysozyme added
D2	B. subtilis
X	Unknown, lysozyme added
Y	Unknown, lysozyme added

Table 2: Shows what species of bacteria was in each letter-coded well.

Discussion

In this experiment, seven bacterial lysates and two unknowns were ran through electrophoresis in order to determine the banning pattern or each and determine the identities of Unknown X and Unknown Y. After electrophoresis, the gels were then dyed using the organic stain, Coomassie Blue. The Coomassie dye molecules bind the amino groups of proteins by ionic bonds allowing bands to appear at the different protein molecular weights. To determine the molecular weight of each protein component in each sample, one can use the Rf standard curve, see Figure 2, curve to predict that value of the molecular weight of that specific band. Once the Rf value for a given band is found, the weight can be found using the best fit line. However, one should note that just because the band appears to be the same weight as a standard that it does not mean that is is the same as the standard. If the bands are matching, it only means that the particular protein has the same molecular weight and may actually be a different protein.

The points are suppose to create a best-fit line seen in Figure 2 and from the points plotted, an exponential curve would be formed if all of the points were connected. Possible reasons for this is that there was contamination which causes the wells to show the wrong molecular weight and banning pattern, or if the gel has not been allowed to run fully giving the gel an incomplete banning pattern.

Table 2 shows what species where in each well. If lysozyme was added, then denaturing took place. Lysozyme is an enzyme that is capable of breaking the glycosidic bonds between the N-acetylglucosamine and N-acetylmuramic aid groups that make up the glycan rods of the peptidoglycan. When lysozyme is added, the protein denatured causing the proteins to unfold and to separate out causing multiple bands in the SDS-PAGE. If lysozyme is not added to the species, then no denaturing took place meaning that the protein did not unfold and few, if any, bands would be seen in the polyacrylamide gel.

In Figure 1, the wells that did not have lysozyme added (A2, C2, D2) only showed a few bands, if any, meaning that the because of the lack of lysozyme, the sample did not break down and was too heavy to move through the gel. For the remaining wells that did have lysozyme, it can be seen that there are many more bands, meaning that the lysozyme did break down the protein and caused a separation of the sample’s proteins. Each protein that made up the sample is shown in the band and can be compared to the standard ran in well “S.”

Comparing A1 to A2 there is little to know difference in the protein profile seen in the SDS-PAGE. The reason is that A1 and A2 both contain E. Coli, which is gram negative. Because E. Coli is gram negative, it lacks a thick peptidoglycan layer and can be lysed by the treatment of SDS alone, it is irrelevant if lysozyme is added. Since it is non-consequential is lysozyme is added, both profiles or A1 and A2 should be the same.

Comparing wells C1 and D1 to C2 and D2, C2 and D2 did not have lysozyme added and therefore there were only a few bands appeared when compared to C1 and D1. The lysozyme was the key in unraveling the sample, and because it was not present in C2 and D2, there were almost no bands. From this, one can conclude that M. luteus and B. subtilis are gram positive and have a thick peptidoglycan layer with just one lipid bilayer.

In order to determine the identity of the unknowns, the banning pattern is

compared to that of each of the known sample. Whichever sample that most resembles the unknowns most likely are the identities. Unknown X is most likely E.Coli. When comparing well X to A1 and A2, the banning patterns looks almost identical. Unknown Y has a banning pattern most resembling well D, B. subtilis. When comparing the unknowns, it is possible to come to the wrong conclusion if the gel has not ran to full completion, if there was a switch in the identities of the wells, or if there was contamination in the wells. If there was a contamination in the wells or if the sample ran was not pure, then there would be extraneous bands in the sample causing confusion into unknown’s identity. Another possible problem in the gel is for smearing to occur. This happens either by contamination or the sample having too many proteins that are of similar weight so that there is a consecutive run of bands depicting as a smear.