I performed protein estimation using BCA FROM thermo-scientific,i did dilution 1:10,1:100,1:1000. i noticed that the three results after i multiply by dilution factor is very far from each other,or sometimes only one dilution is working and the other in negative value.What could be the problem and what is the action in this case?
note:
if this is a case of sample, then either you have thiol or more concentrate proteins. Every methods have some linear range and by the different dilutions, we get idea about the linear range for our sample. for ex. if you did 10 times dilution and OD is not the range of std, it will give false estimation. Please try to make dilution enough so your samples od falls the middle range of std like if you started from std 25 to 1500, your OD almost falls around equal to 600 std. then you will get best results.
You have to use a correct blank-baseline, for each dilution. If you are using the same amount of solvent in each dilution, then with the same blank is not possible to have negative values. I will need more details about what are you doing and then I could help you better.
You should have at least very similar results in terms of concentration.
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