SYPRO RUBY protein blot staining and Subsequent Western blot - (Apr/24/2008 )

Hi everyone,



I am working on the GABAA receptor protein and analysing the protein expression of these receptor proteins using Western blot. I use actin as a housekeeping gene, but am currently trying out the SYPRO RUBY blot stain, which I also intent to use as normalization for variation in loading between lanes. I have several questions with regards to this...

Firstly, my first few results showed a marked reduction (at least 10X) in band signal intensity of the blot that went through the SYPRO ruby staining steps when compared to the ones that did not... has anyone who has used SYPRO ruby blot stain excounter such an effect.

Another question is, if staining with SYPRO ruby proves to cause a downstream immunoblot problem, would it be possible to first proceed with immunobloting and subsequently strip the membrane and stain with SYPRO ruby. Can anyone confirm if stripping actually removed the block as well?

I am also worried about the effect of the fixing step before staining with SYPRO RUBY... using 7% acetic acid and 10% methanol. I was told that methanol is quick difficult to be removed and hence may cause reduced antibody binding, explaining the reduction in signal intensity as seen in my case. Can anyone explain the purpose of fixing prior to staining or can I actually omit this fixing step...

Please help! Am really at a lost here.....

THANKS!!

Sindy

I don't think you can use Sypro Ruby to stain a membrane. I think it's only meant as a total gel stain. From what I understand you're looking for a loading control. What's wrong with stripping and reprobing for actin? If they are all loaded the same then you should have the same ammount of actin in each well. If that doesn't work then you can maybe try another loading control such as alpha-lactalbumin.

Anyway as far as membrain stains go try Pierce's MemCode reversible protein stain http://www.piercenet.com/products/browse.c...WT.mc_id=keynum. I've tried it and it works pretty well to stain the entire membrane for proteins.

Hi Karate Kat,

Thank you for your suggestion. There is in fact, a Sypro Ruby protein blot stain that is used specifically for protein blots, available from either Molecular Probes or Lanza, which I am currently using but giving me some problem.

Yes, I am looking for a loading control and yes, I am also using actin as a housekeeping gene, however, in the animal model that I am working with, which lacks the protein dystrophin, questions has been raised as to if actin expression is perturbed. This is because actin binds to Dystrophin and also, the expression of several other proteins which is associated with dystrophin has been found to be reduced in this animal. Question has been raised too as to if other common housekeeping gene used for brain such as GAPDH is altered as the metabolic functions in these animals are effected. Which is why I am trying to use the most old fashion way of loading control, i.e. either a sister gel but prefer a stain use to reversibly stain blots post transfer.

ALso, the protein of interest that I am currently analysing is very sensitive and also some of the fixation steps used in the Sypro ruby has caused a significant reduction in signal, the pierce product that you suggested which doesn't involve these steps sounds promising. However, I will need to clarify the content first with the company to see if it contain anything that will interfere with subsequent downstream immunoblotting. Just out of curiousity, have you compared blot treated with it versus ones untreated?

SIndy

I am also eager to use the Sypro ruby blot stain to quantify the total amount of protein transferred to nitrocellulose membranes. I also found that membranes stained with Sypro ruby before blotting wiped out the signal seen for blots processed normally.

Did you find out anything from Invitrogen or Molecular Probes? Did you try out Pierce's MemCode as Karate Kat suggested?

Thanks,
David