We also had a hard time with EGFR at the beggining, but it works when we used overnight transfer at 4oC..

Hi Samantha,

methanol in the transfer buffer promotes dissociation of SDS from the protein

and improves adsorption of proteins onto membranes in the presence of SDS.

First of all, can you use a 4-20% gradient gel? Otherwise, for this size of protein use a lesser acrylamide concentration. Regarding transfer: if your smaller size protein and/or your loading control looks good, then it is likely that transfer of the large protein is the problem. If you get variable transfer between blots using the same lysate then it is transfer problem most likely. If you only have trouble once you use a new lysate then perhaps you do not have enough of target protein. So try using the same lysate on separate occasions. To enhance transfer from the gel, you could use less methanol. In my experience from teaching students, often people do not observe the length of time the gel is sitting in the transfer buffer prior to transfer. So, if you need to leave the gel in the buffer for 20minute, then make sure that you only go over this time by 1 max 2 minutes, as methanol can make the pores smaller, hindering large MW transfer. Also, ensure that when you wet your membrane, you shake the dish once you put in the membrane so that the membrane wets evenly.

You could also stain you gel after transfer to see how much protein remains in the gel. I can not comment on transfer conditions as we use semi-dry.

I think the PVDF also needs to be wetted by methanol before use.

Yes, I do reconstituted my PVDF membrane in methanol prior to soaking it in transfer buffer. I tend to let it sit on the rocker in transfer buffer for 10 minutes prior to building my sandwich.

I soak my gel in non-methanol 1x transfer buffer to avoid dehydrating the gel and to remove some of the SDS from the gel. However, because I am thinking it is a transfer problem of my large protein, I did not think excess SDS from the gel was such a problem, so I have not been soaking the gel in transfer buffer for very long prior to sandwich.

I could try a 6% percentage gel, but I would rather stay in this range as there are 40kD proteins I am also interested in and do not want to run off the gel. Sadly, I do not have gradient gels.

For big proteins (eg glycosylated receptors) I use the 37,5:1 acrylamide solution and blot (onto nitrocellulose) ON at 35 V. Works well for the leptin receptor (150 kDa) ...

Try using precast gels (bio-rad), 4-20% gradient, and add 0.05% SDS to your transfer buffer.

I do transfer @ 100V (constant) for 75-80mins at 4C for ACC (250 kDa) and it works fine.

You can also change your lysis buffer to RIPA though that shouldn't be the issue I feel (from my experience working with IR, RON and other growth factor receptors)....

good luck...

Hi Samantha,

We routinely blot for EGFR in multiple cell types. Our lysis conditions are as follows:

Harvest cells on ice in cold pH 7.5 lysis buffer (20 mM Tris-HCl , 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 50 mM sodium fluoride, 1 mM Na3VO4, 1% Triton-X100, 1 mM DTT) supplemented with protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). After normalization of concentration, samples are diluted with Laemmli buffer and denatured by boiling.

We use gradient (4-10%) or straight 8% acrylamide gels for EGFR, depending on additional proteins of interest. We wet transfer to nitrocellulose membranes. PVDF should also work, but we prefer nitrocellulose for larger proteins and only use PVDF for low molecular weight (up to 30 kDa) proteins.

If you can detect Akt then I don't think there will be any problem with lysis because EGFR is a cell surface protein and can be lysed easily?

(1) Total Akt is abundantly expressed in cells and so can easily be detected, however, the cells you are using might have low EGFR expression level so you may need extended exposure of the membrane for detection in dark or alternatively load more protein during PAGE.

(2) The EGFR antibody which you are using can be of poor quality, if not tested in your earlier experiments.

(3) Keep the same 8% gel and increase transfer time to 3h or do transfer at 100 V for 2h or 80V for 2.5h.

I usually detect 165kDa protein with 8% gel, and keep transfer time 2.5h at 80V (wet transfer at 4C). PVDF or NC membranes should not be a problem (I guess)....

Good luck

Hi Samantha,

You should try to stimulate with EGF and or HGF to activate and increase your EGFR expression. You can also try to IP EGFR, which works fine in our lab, even though the protein is not expressed without IP, because the load is to low (5*10^6 cells will be enough). We always use antibodies from cell signaling. In our hands, the method of cell lysis is not that exclusive for a proper westernblot, but mainly the protein load. Good luck!! Kind regards, Kim

Thank you for all the suggestions! Lots to consider.

I am currently trying a longer overnight transfer with a nitrocellulose membrane. If this does not work I will consider my lysis conditions. Thank you again for the suggestions!

Use SDS in your transfer buffer (Check bio-rad website for TGS10X buffer).

After transfer, wash with water several times and stain with Ponceau Red to check the transfer efficiency.

Good luck

You can confirm your lysis condition very simply....

Spin down cells in two different tubes. Dissolve one of the cells pellet in your lysis buffer (sample 1) and the other directly in protein sample buffer (sample 2) and then proceed as usual. Keep the transfer condition same. If you detect EGFR in sample 2 but not in sample 1 then it definitely means there is some problem in your lysis buffer. However, if you can not see the EGFR protein band in both of your samples then problem is somewhere else, (most probably in the expression level or transfer conditions)....

I used to do western blots for egf receptor. I don't remember any particular issues with this protein. I would maybe transfer longer though. If your heavy molecular weight marker (250kd) is totally transferred onto the membrane(nothing left in the gel), then the egf receptor should also be there. If the transfer worked for the heavy proteins, the problem is likely the antibody. But you could run the insoluble portion of your lysate also to be sure. This will dissolve in the sds loading buffer with boiling.

We also had a hard time with EGFR at the beggining, but it works when we used overnight transfer at 4oC..

hi samantha how are you?hope you are doing great

can you try 30V for overnight incubation for transferring protein? just try it and tell me ok hope it will work. all the best

Hi Samantha, just simple suggestion try two times transfer buffer under the same conditions you mentioned above. This may work most probably, considering the higher molecular weight of the protien. Good luck !!

We also routinely transfer at 25V overnight and have no trouble detecting EGFR. There are some cancer cell lines with very high EGFR expression eg MDA468, A431 for use as a positive control. Good luck!

Use 0.25% of SDS in your transfer buffer & run at 250 mA for 90 minutes. SDS enhance the transfer of big proteins above 60 kDa molecular weight. I am usually able to transfer 250 kDa proteins by this method.

I had very good results transfering RPTK like EGFR by transfering on PVDF in complete absence of SDS which is not so good for that. Transfer buffer is composed of 25 mM Tris, 200 mM Glycine and 20% EtOH. Transfer is done for 2 hours at constant amperage 200 mA or o/n 30 mA in a cold room. After transfer membrane is rince in TBST, stained with Coomassie, destained and satured with whatever you like.

Concerning your lysis conditions it should be fine for solubilizing EGFR, despite the fact I can't see the point of sonicating except loosing materials. Hope this will help.

@ Botond Cseh : Thanks for your information I am using Hybond C membrane and 0.25% SDS, it is working very perfect for me.

Definitely try wet overnight transfer. Although semi-dry also works, but most consistent results are obtained with wet overnight transfer. Use nitrocellulose membrane. You use the same lysis buffer as me (I have 150 mM NaCl in addition, PMID: 19003995). Always works.

use tris acetate 3-8% precast gels and transferring in a low A for several hours in cold room.I have used this not on this protein but other one more than 250kD.transfer was ok

Hi Samantha,

Have you figured out what was the problem with your transfer?

I'm having similar problem and I would be happy to hear from you if you found a solution.

Hi Viviane

may be this is late but still I would write some suggestion....

I presume that the problem Samantha was facing and as you too are asking could be bacuse of the precipitataion of the membrane part due to high speed centrifigation- this method obviously works for whole cells lysate and for membrane proteins expressed in higher concentrations. But if the concentration of your membrane is low, it won't be detected while you develop your membrane! So the better way I would suggest to lyse your sample in lamelli buffer and sonicate for 3-4 cycles. Also to avoid precipitation while boiling, you could heat you sample at 37-40 degrees / 30 minutes instead. hope this helps

good luck