I run the sds page but after transfer to membran and staining I saw that lower band are biger than higher band. How can i solve it?
You need to give sufficient information to get relevant answers, comments and suggestions that will be helpful to fix your problem.
Did you load cell lysates or purified proteins on your gel? If you load cell lysates, you need to resuspend the cells uniformly.
If they are purified proteins, how did you purify the proteins? Did you use high concentration of salts in your purification buffer? Did you quantify your total proteins? How much proteins did you load on your gel? What is the size of your target protein? how did you prepare your samples for SDS-PAGE analysis? Did your sample buffer contain appropriate amount of detergents?
I am speculating that this problem might happen either due to overloading of the proteins or due to the presence of high salt concentration in your buffer.
Thank you.
I prepared all buffer based on the protein methods book.
I used the cell lysate. And after Branford assay I loaded 50 micrograms.
Which buffer must be changed, loading or tank buffer?
Did you all the total fraction or the soluble fraction of the cell lysate? 50 ug looks to much. Try to load 2-10 ug and see the result. You can also use fresh sample and tank buffer. I hope it works. Good Luck!
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