Coomassie blue before protein transfer - (Oct/26/2009 )
Is it possible to stain my gel with coomassie blue to see if there are proteins present before transferring onto the PVDF membrane for western blot? Will it have any effect on the antibody association?
Thank you
britkid79 on Oct 26 2009, 06:25 AM said:
Thank you
Yes you can. Don't know of any effects.
fishdoc on Oct 26 2009, 08:17 AM said:
britkid79 on Oct 26 2009, 06:25 AM said:
Thank you
Yes you can. Don't know of any effects.
other than the transfer and capture of the dye by the membrane. you will not be able to develop with color deposition. you will need to do chemiluminescence or fluorescence detection.
destaining coomassie on membranes is difficult and will often interfere with antibody binding.
Moreover teh transfer efficiency will also decrease as your protein is fixed to the membrane (Remember coomassie stain is disolved in acetic acid:methanol:water mix)
Actually once I tried to do a coomasie before transfering and it was a disaster, because the preoteins were already fixed in the gel and I could not get any transfer. So, I would say you can not do it, at least not if you use the normal coomasie and then you destain with methanol and acetic acid.
laurequillo on Oct 27 2009, 05:56 AM said:
I've done 5 or 6 western blots in the past 2 or 3 weeks, each time I stained with coomassie and transferred the protein to PVDF with the Invitrogen iBlot dry transfer system. I was able to detect Flag-tagged antigen on the membranes. However, the maximum I was able to detect thus far was 10 ng of protein. Perhaps some of the protein did not transfer, resulting in me not being able to detect lower protein amounts. However, if low level detection of protein is not required, transerring after coomassie can definitely be done.
I've done 5 or 6 western blots in the past 2 or 3 weeks, each time I stained with coomassie and transferred the protein to PVDF with the Invitrogen iBlot dry transfer system. I was able to detect Flag-tagged antigen on the membranes. However, the maximum I was able to detect thus far was 10 ng of protein. Perhaps some of the protein did not transfer, resulting in me not being able to detect lower protein amounts. However, if low level detection of protein is not required, transerring after coomassie can definitely be done.
