ErbB4 phosphorylation

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Arun Vaidyanath
Arun Vaidyanath's picture
ErbB4 phosphorylation

Hi everybody
Can anybody help me with the phosphorylation of ErbB4 with Western blot. I used the p-ErbB4 antibody from santa cruz biotech but there were lots of nonspecific bands coming out. Is there any internalization of these receptors upon binding? i would like to ask whether we have to lyse the cells  in 4 degrees to reduce this internalizaton? The bands are faint. So if I immunoprecipitate the ErbB4 , will the internalized receptor give signal? And if coimmunoprecipitated can i use any phospho tyrosine antibody (like pY20 or p-Tyr-100) for the band detection?  I hope my question is clear. Thanks in advance.

Chin Fen Teo
Chin Fen Teo's picture
 Hi Vaidyanath,

 Hi Vaidyanath,

Sometime it is unavoidable to get non-specific bands on your westerns... As for your case, the best way to tell is to compare untreated vs treated cell lysates... If those non-specific bands only show up in your treated samples, then you may think they come from the internalization issue. If those bands also show up in the untreated sample, then you may ignore them and focus on the corrected/predicted protein Mw band.

Yes, lyse the cells at 4 degree helps a lot to minimize unwanted events that occur in the cells. Usually, I prechilled every things (wash buffer, lysis buffer, even tubes that I am going to use to put my lysates).

As for the final point, yes, you may do the IP in both ways indeed. (IP P-Tyr/WB ErbB4 or IP ErbB4/ WB P-Tyr)

I hope this helps and good luck. 

Arun Vaidyanath
Arun Vaidyanath's picture
 Hi Pippuri,

 Hi Pippuri,
Thank you very much for your timely reply.... I will take into consideration of your valuable advice. Once again thanks a ton!