Hey Nihad, what exactly do you mean saying "EGFR is a native protein, it does not need SDS"? Why can't you use a normal SDS-PAGE approach? Do you isolate proteins from cells? 

Hi Nihad,

I used to blot EGFR several times.

You don't need any particular condition, except the low percentage of your separating buffer. Don't put SDS in your transfer buffer, it is useless. Just don't forgot ethanol or methanol.

I personnaly use home made gradient gel but it is useful only if you have to check for small proteins at the same time.

I recommand you the CST antibody (rab monoclonal), and for the size it's 175.

Good luck

Thank you all for your kindness and cooperation

Could you David send to me your protocol that you consider it perfect for epidermal growth factor receptor which it is a large non reducing and native protein (170 Kd), including components of both stacking and resolving gels, transfer buffer.

I appreciate your attention 

4X lower tris (separating) : 1.5M tris pH8.8, 0.4%SDS, dH2O

4X upper tris (stacking) : 0.5M tris pH6.8, 0.4%SDS, dH2O

Running buffer : Tris Glycin 25mM, SDS0.1%, dH20

Transfer buffer : Tris Glycin 25mM, Ethanol 10%, dH20

These are actually quite standard conditions.

If you just have to blot EGFR, run a 7 or 8% acrylamide gel.

I would like to mention that "non reducing and native protein" means nothing in western blot, since all proteins have to be denatured at 95° in Laemmli buffer containing B-mercaptoethanol, SDS and DTT. Otherwise, we would talk about a "non-denaturing gel", which is quite different.

Just don't transfer less than 2hrs, since EGFR is a big protein.

Good luck !

EGFR in order to be detected by western blot needs to be separated as a whole non denatured protein so antibody can bind and then can be detected, so SDS can not be used  or even beta mercaptoethanol, excess heat can cause denaturing the protein.

I did it but with transferring time and voltage 15 hrs, 30v respectively but with a weak yeild.

So if you please give an advice from those who succeeded in blotting this protein

Thanks again 

Hi Nihad,

Im sorry to insist, but believe me you are wrong on this.

You will find example of nice WB of EGFR in Ardito CM et al. Cancer Cell 2012.

The basis of western blotting is denaturation.

If you apply my advice you will have nice results.

Cheers.

Thank u all I did it, without any SDS , because my antibody  specific for  non denaturing protein, it was along journey.