problem on western blot - (Jun/08/2007 )

Hi All,

I am trying to detect the expression of EGFR in MDA-MB-435 cell line and unfortunately i am not able to detect its expression at all, some previous work done on these cells shows significant levels of EGFR in them, my control experiment for detection of beta-actin is able to detect the expression of actin. i dont know what is going wrong with regards to EGFR as i have tried a range of antibody concentrations (the primary antibody is a monoclonal anti EGFR from sigma) as mentioned in the manfacuturer's protocol as well as in other papers. my detection system is the bio-rad opti-cn substarate kit and i dont know if this is the problem? or if it is anything else.

any help in this regard is highly appreciated

You might ask them what detection system they used to optimize their recommended western antibody concentrations. They might have used a more sensitive detection method, and that would mean that you would need to increase your antibody concentration. Or you could try adding more sample.

think about some basic questions..

How do you know that your protein is expressing??? okie lets say its expressing.. then wot?

are you want to detect your protein?? what Abs?? n What should be the control?? what about your control??

if your control is working fine.. then u don't have expression... or if your control is not working... then your Abs are gone off...

you can first test your protein expression either by dot blot or ELISA..

all the best..
Amit

semi dry blotting has some limitations, I think with 2h blotting you are working at the border of efficacy;

I recommend to do O.N. tank blotting; we use it routinely to transfer GFP-tagged RTK´s which are nearly 200 kDa large: we also have poor blot efficacy in semi dry but good in tank blotting

Hi,

Maybe you are not using enough protein. Are you enriching for membrane-bound proteins?

I agree with mikew. Do you add detergent to your lysis buffer, such as Triton? If not, you may loose all the membrane-embeded proteins, witch could explain the presence of actin, but not of EGFR.

Also, high molecular weight proteins do not transfer as well as small with a semi-dry transfer machine. But if you use an optimized protocol, you should have no problems with the transfer. I often blot for fatty-acid synthase, with is a little above 200 kDa, and I have a great amount of it.

Cell fractionation is not exactly my expertise, but as suggested above, you need to make sure that your prep aims/includes membrane proteins. A proper control would be an antibody against any abundant membrane protein. This is not only to ensure that your detection system works, but that you include the fraction of interest in the sample. When you said that literature says it's expressed, did they use the same protocol as you did? Because if that claim was based on for example RT-PCR, immunohistochemistry, or confocal imaging it is by no means a given that your WB is going to work before spending some time on figuring out an appropriate fractionation/concentration/detection procedure.

I don't think transfer is the problem. Even if larger proteins transfer somewhat worse (use a gradient gel with low PA content, play with the methanol conc, check transfer efficiency with a prestained ladder) a sensitive detection system such as chemiluminescence will detect proteins that are all but invisible on the membrane. I think that would go for a colorimetric system as well, although the detection limit is inferior.

hi,

you should probably switch to the ecl detection system which is more sensitive to detect smaller amounts of protein in your blots.

greetings
labghost