Hello Maren,

polyclonal antibodies can detect different isoforms of a protein, resulting in various bands. In the case of eGFP this could not happen (hopefully) because there shouldn't be any spliceforms of your fusionprotein, so i would give it a try.

good luck

Hello Simon,

Thanks a lot for the answer.

Maybe I should explain my experiment in more detail: I want to compare eGFP concentrations in different cell lysates to an eGFP Standard on Slot Blot. I wondered, if the higher number of binding sites of the polyclonal antibodies leads to steric hindrance and consequently to impaired linearity at higher eGFP concentrations and if therefore the use of monoclonal antibodies which bind only one epitope is recommended for quantification.

Maybe someone has experiences in Western Blot Quantification and the influence of polyclonal/monoclonal antibodies?!

Thanks.

Hi Maren,

If you have the same epitopes in both your GFP proteins, quantification should be similar regardless of the number of epitopes you are detecting at the same time. If the lysates are similar as well your background binding should be similar too in different samples (thus if you have a quantified standard it would not hurt that it is in a comparable lysate). In any case monoclonal can also bind unspecifically as long as there are other regions similar to the epitope.

WB quantification is not an exact technique anyway, you have other things to worry about depending on the way you detect your WB. chemiluminiscence vs fluorescence or even vs xray