5 replies to this topic

[cedricSZ]

Dear all,

When I performd WB, I have always some variability in my control gene (GAPDH) and this variability seems to be correlated in some part to the variability of the gene of interest (here it is a-SMA staining, see film in attachment). Does anyone have ever seen that before, does anyone can help me. My membrane are pretty clean without background, and the Antibidies used works very well.

Many thanks

Attached Files

•  WB090805_IMR_aSMA_929.bmp   1.98MB   65 downloads

Edited by cedricSZ, 05 August 2009 - 07:47 AM.

[Dr Teeth]

cedricSZ, on Aug 5 2009, 11:22 AM, said:

Dear all,

When I performd WB, I have always some variability in my control gene (GAPDH) and this variability seems to be correlated in some part to the variability of the gene of interest (here it is a-SMA staining, see film in attachment). Does anyone have ever seen that before, does anyone can help me. My membrane are pretty clean without background, and the Antibidies used works very well.

Many thanks

There's no attachment.  Are you loading equal amounts of protein per well?

[cedricSZ]

Dr Teeth, on Aug 5 2009, 05:42 PM, said:

cedricSZ, on Aug 5 2009, 11:22 AM, said:

Dear all,

When I performd WB, I have always some variability in my control gene (GAPDH) and this variability seems to be correlated in some part to the variability of the gene of interest (here it is a-SMA staining, see film in attachment). Does anyone have ever seen that before, does anyone can help me. My membrane are pretty clean without background, and the Antibidies used works very well.

Many thanks

There's no attachment.  Are you loading equal amounts of protein per well?

Dear Dr Teeth,

Sorry for the attachment, now it is done. Yes I loaded an equal amounts of protein per well (5µg)

[gfischer]

The simplest answer is that GAPDH isn't stable across the condition/treatment you're studying.  You may have to look for a different control, like B-actin or one of the tubulins.

[drjcroberts]

hi,

what can happen is variability in transfer of the proteins. but i agree with the statements of others; try a tubulin antibody

you must post-stain your gel with coomasie, if there is any remaining, then you should alter the transfer time or conditions

J

[cedricSZ]

Dear all,

Many thanks for the answer. My cells are treated with TGFb which could probably induced unstability to the GAPDH ????? but I have tryied also with a tubule which was variable also.

First I have to cheek wheter the coomasie staining is OK.
Secondly I have to test a B-actin gene but my gene of interest is a a-SMA (actin gene also) with the same mol W that the B-actin. So I have to strip my membrane.

Does anyone have a good procedure and buffer for the stripping of membrane.

Thanks in advance.