Hello.
I have made a Bradford reagent and i want to know if my standard curve of my reagent is correct.
I have made an standard curve with this concentration of BSA (with the final volume of 0.5ml for each standard solution) respectively: 0.125, 0.250, 0.500, 0.750 and 1 mg/ml but the absorbtion of these standards in 595 nm are 0.178, 0.258, 0.459, 0.640 and 0.842 respectively.
After i used my reagent in order to find out my protein concentration the results made me confuse.
The protein A595nm are mentiond as follow:
Cell Lysate: 0.421 with 4 fold dilution of cell lysate.
Supernatant (Clear Cell Lysate): 0.390 with 4 fold of dilution of Supernatant.
Solublized Inclusion Body:0.174 with 4 fold of dilution of I.B solution.
According to the standard curve of my reagent the amount of protein in each solution(each solution has volume of 30ml) are as below:
Cell Lysate: 1.81 mg/ml * 30ml=54.3 mg
Supernatant: 1.65 mg/ml * 30= 49.53 mg
I.B solution: 0.516 mg/ml * 30 =15.504 mg.
But there is no balance in the amount of proteins i mean 54.3 is not equal with 49.53+15.504.
what is the problem? is that because of my reagent standard curve? did i make the correct standard curve or solution of BSA?
should i solve BSA in buffer? because i have solved BSA in Water.
Thanks for your valuable suggestions.
I can name several factors affecting the results.
1. Different proteins have different affinity towards the dye, thus using BSA to quantify all proteins in the universe is a major approximation
2. Anything that can compete with the dye for protein interaction will do so
3. The method is extremely sensitive to volume variations (it is a pain in the ass)
4. It is not a linear method, no matter what biologists say
Protein quantification using Bradford's method is a terrible quantification procedure, it should be used as an approximation method. A mass balance will never check. (Unless you are quantifying bsa and only bsa is present in your samples in the running buffer).
How did you solubilize the inclusion bodies? This is usually done with a concentrated urea or guanidine HCl solution. These substances will probably interfere with the Bradford assay. You should make a separate standard curve for the solubilized inclusion bodies, using the same solvent as you used for the solubilization.
The inclusion bodies in the total lysate may not register at all in the Bradford assay if they are not dissolved by the Bradford reagent.
If you used detergents in the lysis buffer or to solubilize the inclusion bodies, then you should not use the Bradford assay, which is not compatible with detergents. Use the BCA assay or Lowry assay instead.
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