western clarification - (Dec/16/2005 )

I've been looking for a protocol to do a western blot for EGFR (epidermal growth factor receptor) on rat heart tissue. I've found so many different membrane protein isolation techniques that I've confused myself. Does anyone know a good protocol for this if I am trying to compare expression levels of the EGFR in different tissue samples based on treatment with different compounds?

I was just thinking of possibly grinding the frozen tissue in some Tris-HCl (+protease inhibs +EDTA) , spinning (what's a good approximate speed, I see a lot of different speeds listed), washing again once and then respending the pellet in Tris-HCl + SDS or some other detergent (CHAPs etc..) for blotting.

Has anyone tried any other methods, perhaps something simpler (just lyse in SDS/Triton and run gel?) ?...or do I need to do complex membrane prep steps first?

Any clarification or suggestions appreciated,
thanks

Hello-

There are lots of protocols, but dont worry, protein extraction for western blotting is easy. We usually just lyse cultured cells in cold lysis buffer. If the cells are adherent you have to scrape a bit. If you're extracting from an animal you obviously have to grind a bit. But basically, we just lyse the cells in a lysis buffer containing detergent ( a good starting point is 1% TritonX100), several kinds of protease inhibitors (we just buy the tablets), and if preserving the phosphorylation state of your proteins for western is important you need to add some phosphatase inhibitors. We use 1mM sodium orthovanadate, and/or 10mM NaF. I keep a stock of detergent containing pH 7.4 Tris buffer + 150mM NaCl, and add the proteasse inhibitors and/or phosphatase inhibitor just before use.

After lysis I usually let the lysates rest on ice for about 20 minutes to let everything solubilise, and then spin at max speed in the microfuge for 15 mins. This removes insoluble, membrane bits and cytoskeleton that the detergent can't solubilise. The supernatants are then ready for western blotting.

So the setup is pretty simple. There are lots of different protocols, but lysis in tritonX with inhibitors and a quick spin is all you should need for most applications.

Good Luck,

Mountainman

Thanks for the tip. I'll try that and refine if necessary. I was hoping it wouldn't be too time consuming so I hope this works. I'll try to post results when I finish. Right now I'm just looking at the EGFR expression levels but I also purchased some anti-P Tyr EGFR antibodies as well. We may be looking at the phoshorylation state next so I'll try those other suggestions later as well.

Thanks again and I'll try to follow up if anyone else is interested in similar techniques

It was succesful. They had some stock lysis buffer (HEPES, NaCl MgCl EGTA glycerol and 1% tritonX-100). The only little problem I had was that I detected extra bands in lanes that were supposed to be empty...perhaps just from accidental spillover when loading the gels?? The 7.5% gel also stuck a little too well to the membrane. I had to scrape it off. Other than that it went well, just have to do one more blot and then the final data analysis.