My r-protein was 1.1 mg/ml and gives smear during SDS I decided to use Triton x-100 to solubilize my protein, but the problem is that the conc. increased from 1.1 to 4 mg/ml is that logic
Just by addition of Triton X 100 the concentration of your recombinant protein cannot increase from 1.1mg/ml to 4 mg/ml. You need to find out whether the concentration of Triton X 100 that you are using is not interfering in the protein estimation method. Please ensure the same.
Thanks Deepti , I think you are right , detergents may cause the pedestal surfaces to become “unconditioned” and this may be the reason.
Haa there are many detergents which interfere in protein estimation... Therefore there are many companies like biorad who prepare detergent compatible, reducing agent compatible protein estimation kit (RC, DC BCA kits)
Haa there are many detergents which interfere in protein estimation... Therefore there are many companies like biorad who prepare detergent compatible, reducing agent compatible protein estimation kit (RC, DC BCA kits)
It is not logic! Probably your method interfered with the detergent (mostly do). How much protein did you load in a well, isn`t to much? Do you have enough SDS added? The SDS should be much stronger than triton at much lower concentrations! You can increase the SDS and DTT concentrations and the boiling time. If this does not solve the problem, then there are other issues. The smear may have different reasons such as crosslinking or proteolitic digestion, depends on the position, above or bellow the main band.
Dear Augustin C. Mot, I just got a smear in my last SDS-PAGE and so I added some Triton X-100 to my sample to clear the suspected polymer and after adding this detergent, I measured my protein conc. using Nanodrop as a routine work and I found that protein conc. went up and as you said this is not logic, I searched and found that Nanodrop sometimes gives you incorrect reads out if your samples contain detergent with which formation of sample column during measurement may be difficult.
Triton X-100 has aromatic ring in its structure and therefore it would definitely interfere at 280 nm (like aromatic amino acids such as tyrosine, tryptophan and phenylalanine) therefore in order to get the correct protein concentration you need to resuspend the Triton X 100 in the buffer devoid of protein and first autozero and then take the reading of the protein solution containing Triton X 100. This would surely solve your problem.
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