9 replies to this topic

[britkid79]

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

[fishdoc]

britkid79, on Oct 26 2009, 06:25 AM, said:

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

Yes you can. Don't know of any effects.

[mdfenko]

fishdoc, on Oct 26 2009, 08:17 AM, said:

britkid79, on Oct 26 2009, 06:25 AM, said:

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

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.

[bob1]

destaining coomassie on membranes is difficult and will often interfere with antibody binding.

[Prep!]

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)

[laurequillo]

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.

[fishdoc]

laurequillo, on Oct 27 2009, 05:56 AM, said:

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.

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.

[laurequillo]

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.
[/quote]


Well, It is good to know. Maybe it was because of my staining/destaining method (with methanol and acetic acid) that the proteins were fixed. What staining/destainig solutions did you use?

[fishdoc]

laurequillo, on Oct 27 2009, 08:03 AM, said:

Well, It is good to know. Maybe it was because of my staining/destaining method (with methanol and acetic acid) that the proteins were fixed. What staining/destainig solutions did you use?

Fixed in 50% MeOH / 10% acetic acid for 30-60 min, stained in coomassie 20-60 min, then destained in 50%/10% for at least a couple hours.

Here is a report verifying coomassie stained proteins can be used for westerns: http://www.sciencedi...45154b04d445c2f

But again, the sensitivity could be affected.

Edited by fishdoc, 27 October 2009 - 05:54 AM.

[laurequillo]

fishdoc, on Oct 27 2009, 02:53 PM, said:

laurequillo, on Oct 27 2009, 08:03 AM, said:

Well, It is good to know. Maybe it was because of my staining/destaining method (with methanol and acetic acid) that the proteins were fixed. What staining/destainig solutions did you use?

Fixed in 50% MeOH / 10% acetic acid for 30-60 min, stained in coomassie 20-60 min, then destained in 50%/10% for at least a couple hours.

Here is a report verifying coomassie stained proteins can be used for westerns: http://www.sciencedi...45154b04d445c2f

But again, the sensitivity could be affected.

Very interesting. Thanks a lot. It is always good to learn new things!