6 replies to this topic

[jangajarn]

Hey guys,
     So it was my first time doing a WB. Since I had to rush to an important thing, I did not have time to develop chromogen after 2ndry antibody. I was told that its OK to dry the membrane and later develop color, which I did. We then found out that nobody has ever done that and people dry either before they start the antibody washes or once its completely done, not in the middle. Is there anyway to salvage the situation? People told to try "activate" the membrane with methanol, wash it couple of times with PBS/TBS and then try developing color. But I wanted to ask the community if anybody has done that. By the way I'm using PVDF membrane.
Thanks in advance for your tips and assistance!

Edited by jangajarn, 02 November 2009 - 02:14 PM.

[Prep!]

well i ve never done it this way but i thinkn u can wash it with your wash buffer briefly to wet the membrane and then add secondary one more time and then rewash and develop just to make sure... or alternatively strip the entire membrane for any antibody and start all over again...
Methanol for activation is usually done before the blot.. not sure if u can do it at this point!!
Best Luck

[Gerard]

I have not done it with a primary/secondary couple but with a HRP-coupled antibody where the substrate was gone, I dried the membrane and after 4 for days when the substrate came in, I incubated the membrane in PBS/tween 20 0.05% for 30 minutes and finished the protocol. There was no visible problem with the blot.

[jangajarn]

Gerard, on Nov 3 2009, 12:39 AM, said:

I have not done it with a primary/secondary couple but with a HRP-coupled antibody where the substrate was gone, I dried the membrane and after 4 for days when the substrate came in, I incubated the membrane in PBS/tween 20 0.05% for 30 minutes and finished the protocol. There was no visible problem with the blot.

Thanks Gerard. I'll try that out

[jangajarn]

Just wanted to share what I did: I just wet the PVDF membrane with 100% MeOH for 3 mins and then washed with PBS+tween for 15 mins. The chromogen works perfectly fine. Just to warn, it does not work if you directly wash with PBS+tween and try substrate.. methanol is required.

[Feelcontraire]

jangajarn, on Nov 2 2009, 02:12 PM, said:

Hey guys,
     So it was my first time doing a WB. Since I had to rush to an important thing, I did not have time to develop chromogen after 2ndry antibody. I was told that its OK to dry the membrane and later develop color, which I did. We then found out that nobody has ever done that and people dry either before they start the antibody washes or once its completely done, not in the middle. Is there anyway to salvage the situation? People told to try "activate" the membrane with methanol, wash it couple of times with PBS/TBS and then try developing color. But I wanted to ask the community if anybody has done that. By the way I'm using PVDF membrane.
Thanks in advance for your tips and assistance!

Yep, in fact HRP lives better dry than in water.

[medchemgirl]

If a PVDF gets dried you'll have to pre wet it with MetOH to rehydrate it again because it is hydrophobic and will not wet with buffer.

Pradeep Iyer, on Nov 2 2009, 11:38 PM, said:

well i ve never done it this way but i thinkn u can wash it with your wash buffer briefly to wet the membrane and then add secondary one more time and then rewash and develop just to make sure... or alternatively strip the entire membrane for any antibody and start all over again...
Methanol for activation is usually done before the blot.. not sure if u can do it at this point!!
Best Luck