Anyone have a good western blot protocol? I was planning to run a real-time polymerase chain reaction but the capillaries in the PCR machine were contaminated. Now I cannot run the RT-PCR, which is the best quantitative method of PCR. So i have to resort to a western blot but i dont have a protocol. Can anyone give me the link to a good, reliable western blot protocol?

what supplier are you looking at?

How the hell are the capillaries in the PCR machine contaminated, you should be running closed tubes.

 

As for the western, it's monkey buisness. The only things you need to work out are the antibody specificity and optimum concentrations. The only other thing that can really give you problems is using crappy/old developer.

 

Western's really shouldn't serve as a replacement for realtime pcr consideirng they look at completely different things. Westerns are also qualitative generally speaking unless you know the antibody binding affinity (and have fun with that).

Try any of these: (I'm not sure what details you already know, but these should be helpful)

 

http://www.protocol-online.org/prot/Molecular_Biology/Protein/Western_Blotting/

 

http://www.sgul.ac.uk/depts/immunology/~dash/group/western.html

 

http://www.assaydesigns.com/technical/protocols_western_blot.htm

I, too, am curious how your RT-PCR capillaries got contaminated and why it's impossible for you to get new ones. I've only done Rt-PCR once, but I'm sure the capallaries are plastic and easily replacable... why can't you get new capallaries and make more RT-PCR mastermix for your DNA sample... If you already have a machine to use, the rest is fairly cheap.