While running SDS PAGE for my protein sample the run always stop 3-4 cm above the bottom line and and the separation distance between different band is very close. While in paper for the separation of same samples are quite good. I dont understand the reason may be gel concentration or what? I am using 13% concentration of running gel and 5% concentration of stacking gel.
Hi Shah,
I think you should check out the following questions to evaluate the resolution of your protein on SDS-PAGE.Separation distance of different band is based on the resolution of your protein.
1.Did you try to optimize the % of acrylamide(resolving and stacking gel)based on the molecular weight of your protein? Because acrylamide % in SDS-PAGE is dependent on the size of the target protein in the sample.
2.What is the size of your protein(MW) before using that % of acrylamide?
3.What is your running condition such as voltage(e.g 120V for 1hr is ok for 12% resolving gel).NB-resolving gel possessing higher % of acyrlamide will take longer time for the dye front to reach the bottom.
I hope after you answer them and check again you will be able to get a way out.
All the best!
Have you checked your running buffer? Sometimes the running buffer bleads out of ions. Without ions the proteins can not be pushed further in the electric field.
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