I am trying to my crude enzymes protein sample in SDS PAGE but protein marker (3.5 to 205 kda) band was came while my crude enzymes and BSA 1000 ppm sample was not came the band.
Where, I am using 12 % and 10 % Gel and also using staining of Brilliant Blue R 250 and silver staining.
Sample preparation
4 ml D/W, 0.5 M Tris HCL 1ml , 10% SDS 1.6 ml, 0.4 ml Merceptoethanol, 0.05% Brilliant blue R 250, glycerol 0.8 ml
total 8ml
I think the problem is that your protein samples are too dilute. Have you measured the protein concentration in the sample before diluting it with sample buffer?
Dear Balvindra,
What I feel the problem is with your sample preparation. Maybe it was too diluted with loading dye and uneven amount of protein was loaded in the wells. or maybe abnormal heating of your sample before loading. These are the probable reasons for getting smears in the gel. I have also faced the same issues.
Suggestions:
1. Quantify the protein for each samples and load uniformly.
2. Heat the samples with loading dye for 95 degree C for 3 minutes. Make sure that the tubes are properly sealed while heating otherwise your sample may dilute again.
Hope this will help you.
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