I am doing an experiment on proteins. I precipitated protein using TCA- acetone method and want to check purity on SDS-PAGE. I used 25-30 microgram protein for one well, after running SDS- PAGE, stained with Comassie brilliant blue and when I destained , no bands appeared other than marker and BSA protein on gel. how can I resolve this problem?
Hi Reena,
It could be that your proteins were eluted from the gel? It is a possibility. If this had occurred, you can try treating the gel with a fixer solution before Coomassie staining. The solution I use is [3.5 % sulfosalicylic acid, 11.5% trichloroacetic acid (TCA)] for 30 min, and then remove solution and perform 4 washes the gel with distilled water for 5 min each.
Hello Reena,
Prior to loading on SDS-PAGE, which quantitation technique did you use? Techniques such as Bradford are less reliable. Our team ( Ravish Godse and me) uses BCA assay to quantitate the protein obtained. On completing BCA quantitation, you can try loading only 10 microgram of your protein on SDS-PAGE and do CBB staining. In terms of the staining technique, it is dependent on the quantity of protein you want to visualize. If this does not work, I would suggest you try silver staining since it is more sensitive in comparison to that of CBB.
How do you know you used 25-30 ug of protein? How did you calculate that. Also is it a mixture of differently sized proteins or 1 purified protein?
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