I did bradford assay and I want to calculate the amount of soluble protein in my glycoprotein sample.
I prepared BSA stock of 1mg/ml and did a standard curve ranging from 0.01 to 0.16 mg/ml.
I got for y = mx + c as y = 3.0669x + 0.0213.
I converted the concentration (x-axis) from mg/ml to mg, hence now ranging from 0.0004 to 0.0064 mg.
I got y = mx + c as y = 76.672x + 0.0213.
I prepared a sample concentration of 1mg/ml (1mg of sample in 1ml of distilled water).
40ul (0.04ml) of the sample was mixed with 160ul of bradford reagent.
I ran my sample in a 96 well plate at 595nm.
The absorbance I got was 1.313 (the abs of my blank is 0.48).
Because it was outside my standard curve range, i diluted my sample by half
(abs of 1:2 = 1.069, abs of 1:4 = 0.767)
The absorbance i got for 4 times dilution is (0.767 - 0.48) 0.287.
If i use the formula = (R) x (TV/SV) x (DF/Wt),
where:
R = protein content from standard curve(mg)
TV = volume of sample prepared from extraction (ml)
SV = Volume of sample used for microplate measurement (ml)
DF = Dilution factor
Wt = Sample weight (mg)
then:
((0.287-0.0213)/76.672) x (1/0.04) x (4/1) = 0.347 * 100 = 34.7%
Is this the correct way to calculate the protein content? If i want to express it as g/100g then how?
Thanks
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