Bradford Assay

m
c
OD595
Sample Volume (ml)
Concentration (mg/ml)

Protocol:

add to a 1ml cuvette:
800 ml PBS
200 ml Bradford Reagent
mix, set absorption at 595 nm to zero

add to a 1ml cuvette:
800 ml - sample volume ml PBS
1-100ml Sample
200 ml Bradford Reagent
mix measure absorption at 595 nm

Defining m & c values:
Measure OD595 for 1,2,5,10,20 mg BSA
Make a plot mg BSA on the x-axis and OD595 on the y axis
Make a linear fit, m is the slope and c the crosspoint with the y axis at 0 mg BSA

Bradford Reagent:
Biorad (store at 4C)

PBS:
0.9% NaCl
0.1 M NaPhosphate buffer pH 7

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Buffer Calculator

Buffer
pKa:Mw(g/mol):
pH
Concentration (M)
Volume (l)
Concentration HCl/NaOH (M)

Recipe:

The amount of titrating agent is calculated using the formula:

pH = pKa + lg([bufferdeprotonated]/[bufferprotonated])

5 M HCl can be made up as follows:
dissolve 491 ml HCl 32 % or 417.5 ml HCl 37% in water up to a final volume of 1 l

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