i am performing an native page of my protein but i am not getting good bands can any one help me to figure it out
Did you extract your protein fresh? it is very important to have fresh protein, ideally the same day extracted protein you should use. What kind of extraction buffer do you use? Do you add protein inhibitors? Importantly, try to run your gel in small milliampere like 15 (15 for one gel). Also, you can try to run different concentration of your protein in one gel.
, then you will have idea which concentration work best for you. I don't know how you made without whole description. But, how my suggestions somehow will help.
Good luck
Praneeth Kumar Killaka
How much concentration of protein you have loaded???
If you didn't calculate the concentration of protein, do check by bradford method and use 30 microgram per microlitre protein... If not try with different concentrations to check which works best for you...
Hello Syed Abrar Qadri,
what is the purpose of your experiment? Always good to know. Generally, you should use some primary and secondary antibodies (western blot) to check you have isolated the protein of your interest. Nonetheless, it seems that you haven't isolated any protein. Did you try to measure the concentration of your protein samples spectroscopically (using e.g. Nanodrop, or some similar device)? Also, you can utilize the Bradford method, as Nishanth Sekar recommends to you. Also, what is the supposed molecular weight of your protein product? If it was p53, it ran out of the gel :)
Hope this help,
Martin
It looks like protein sample is of not good quality or maybe degraded/fragmented into small peptides. If there are proteins then they should appear with sharp bands. The smearing lane is an indication of degraded protein. Try to get the fresh sample of the protein and run in the fresh gel using fresh running buffer.
Try increasing the protein concentration...your protein maybe too diluted and also try a different gel concentration for both stacking and running gel so that the protein are properly place and not smeared. Try keeping the protein away from any contamination from its surrounding as well.....keep giving your ur best...
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