I use 8% gel And 5% stacking gel. I use BSA protein solution diluted into PBS for different concentrations. I ran it for 45 mins at 130V. However, my bands look chubby. How can I fix this? Could it be my acrylamide solution or is it a sample buffer problem?
Does your PBS contain >100 mM NaCl/KCl? I would dilute the protein in buffer with much lower concentration of these salts. High ionic strength usually distorts protein bands.
Were your samples left in the wells for some time (> 5 min) before applying the power? If so, some protein would diffuse into the wells' walls and then would go incorrectly.
There is too much salt. if you use BSA, dilute it in 10-25mM phosphate buffer. if you can use only sample in PBS, i'd precipitate them first.
Try to load a protein ladder to figure out whether it is sample originated or gel originated? If the ladder is ok, then we may discuss your sample and sample buffer ingredients, running buffers and reduction/denaturation processes, gel casting application, etc. By the way, generally speaking, I would prefer a high percentage to capture BSA. 8% is a bit low and convenient for large proteins. In my opinion, 1XPBS or relative ionic strength buffer compositions should not be a problem if you can prepare Leamnli buffer and sample at the correct compositions, if you think you have trouble with PBS (freshly prepared), just perform several dilutions and load each to separate lanes.
Emir
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