western blotting

[Igor-IICB]

Hello everybody who is interested.

I am running multiple westerns using

gels (I would consider small/standard) 55 x 85 mm.

I have just few questions (to start up).

Is there anybody who is in the same technology?

 

Thanks,

 

Igor-IICB

[CharonY]

It is a pretty standard technique. I am pretty sure that me and a couple of others on this board can help you out, if you got specific questions. For broad overviews obviously other sources are obviously better. Bioanalytical and molecular technique textbooks, for example (Sambrook is a standard). Also some manufacturers offer surprisingly nice manuals.

Edited October 31, 2011 by CharonY

[Igor-IICB]

It is a pretty standard technique. I am pretty sure that me and a couple of others on this board can help you out, if you got specific questions. For broad overviews obviously other sources are obviously better. Bioanalytical and molecular technique textbooks, for example (Sambrook is a standard). Also some manufacturers offer surprisingly nice manuals.

 

Dear CharonY,

 

Thanks for your reply. I've done quite a bit of work in molecular biology but I just started Western business.

I am doing membrane stripping today and I could see so many different protocols with regard to this.

Having just few questions, please:

 

1) Some people mention that beta-mercaptoethanol traces may damage antibody (AB) in the future probing

Others seem to be pretty relaxed about this concern. What would be your take on this?

2) I am trying to do 8 (eight) Western gels/membranes at a time. How many of those usually people do?

[CharonY]

Dear CharonY,

 

Thanks for your reply. I've done quite a bit of work in molecular biology but I just started Western business.

I am doing membrane stripping today and I could see so many different protocols with regard to this.

Having just few questions, please:

 

1) Some people mention that beta-mercaptoethanol traces may damage antibody (AB) in the future probing

Others seem to be pretty relaxed about this concern. What would be your take on this?

2) I am trying to do 8 (eight) Western gels/membranes at a time. How many of those usually people do?

 

1) I generally try to keep my samples MS-compatible, so I generally use DTT rather than mercapto for reduction. Generally, I would not expect to have much of it left after the run, though, so it should not interfere too much with immunoassays post-blot.

 

2) That highly depends on the particular instrumental setup you have.