Protein won't transfer for Western Blot
Posted 03 December 2009 - 12:01 PM
I am running a western blot and I can't get my protein to transfer from the gel. I ran the transfer, stained with Ponceau S and saw that my positive control protein and my ladder both transferred but that my protein did not. I then stained my gel and my band was still on the gel. Does anyone have any suggestions or has anyone experienced this same problem?
Posted 03 December 2009 - 12:29 PM
Could you please describe the conditions of your transfer? If you provide more information, we may be able to help you better.
Posted 03 December 2009 - 05:59 PM
Is there anything funky about its sequence or charge?
Posted 03 December 2009 - 06:10 PM
As well, the conditions of my transfer are a wet transfer using nitrocellulose, the buffer is Methanol, Glycine, Tris and Water. I ran the transfer for one hour at 100V.
Posted 03 December 2009 - 07:40 PM
So what i am trying to say is that may be some protein got traasferred but could not pick up the color due to the specificity. So may be its incomplete transfer...
Also this can be possible if you have loaded too much protein amount. I would also be interested in knowing if this is crude protein or the purified one??!!! How have u checked your expression?? cause if its a crude mix, then may be what u are seeing in the gel is some unrelated protein at the same molecular weight!!
What is the percentage of gel tat u are using??
U can also try PVDF once...
Sorry if i have confused you ior asked for too much information?!!!
Edited by Pradeep Iyer, 03 December 2009 - 07:41 PM.