When doing AMS assay, the reduced and oxidized band are not clearly divided on 16% SDS-PAGE tricine gel. The condition of running gel is at 30mA. I want to get more clearly divided bands to do quantitative calculation. The picture for what I did is attached. Thanks.
Dear Yaven
which is the MW of your protein? How many cysteines it contain and you would like to alkylate with AMS?
Because so small difference with a 17% gel look me quite strange if the MW of your protein is not very big. I used it in the past with a proteins with 2 cysteines and a MW of about 20KDa and using a 17% Tris-glycine gel i obtained a more evident separation (see the attached file - suppl fig n°9 from Article Human Sco1 functional studies and pathological implications ...
Of couse as, Jasim suggest you, is it better if you load a smaller amount of sample to obtain more defined thin bands, but i suspect that your bands do not correspond to the protein with and with-out AMS, but to the protein reduced and non reduced and that AMS alkylation for some reason did not work properly.
How do you perform it? in which buffer? which AMS, protein concentrations?
ciao
Manuele
At first, thanks for your advice, Jasim Al-saadi , I'll try. As for my assay, Manuele Martinelli , the MW of the protein is about 23kD with 2 cysteines to be focused. I did the control with and without AMS in buffer mixed with AMS and sample buffer, which showed it worked. The protein concentration is about 20uM.
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