Expression of EGFR in MDA-MB-435 cell line - (Aug/02/2008 )
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. i dont know if this is the problem? or if it is anything else.
any help in this regard is highly appreciated
-bhappy-
QUOTE (bhappy @ Aug 3 2008, 03:49 AM)
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. i dont know if this is the problem? or if it is anything else.
any help in this regard is highly appreciated
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. i dont know if this is the problem? or if it is anything else.
any help in this regard is highly appreciated
A lot of EGFR Abs are rubbish IMO. BD Bioscience I believe is the best one I used to use when looking at EGFR (Western blots). how are you trying to detect? IF or WB or another way?
Stupid question but have to ask - if you are doing WB I assume you have a reducing agent (ie. beta-mercaptoethanol or DTT)? Without one of these in your sample buffer you won't see EGFR at the correct size (~180kDa).
mcl
-mad_cereal_lover-
QUOTE (bhappy @ Aug 2 2008, 06:49 PM)
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. i dont know if this is the problem? or if it is anything else.
any help in this regard is highly appreciated
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. i dont know if this is the problem? or if it is anything else.
any help in this regard is highly appreciated
maybe problem with high molecular mass protein blot transfer? try O.N. tank blotting; what about immunocytochemistry?
-The Bearer-