I am attempting to do a western blot to look for differential MMP7 expression in skin biopsies. I know the expression is differentially regulated based on immunohistochemical evidence and want to further prove it by WB.
My question is, following a bradford assay, how do you know how much protein to load onto your gel in order to get visible bands. How do I know the relative amount of MMP7 in my samples? For example, I know the total protein is at a concentration of 1500 ug/mL, but how can I figure out a good amount of material to run on the gel without wasting the samples (my biopsies are very precious and we don't have a lot troubleshoot with)
Any help is appreciated.
Bonnie
Hi Bonnie,
It depends, how much of your sample do you have? From your Bradford assay you already know how much total protein you have per ul (~1.5ug/ul) so you could load any where from 10ug to 50ug of protein per well. For example, to load 20ug per well, you will need 13ul of your (protein estimated) sample.
In order to know how much MMP7 you may need to perform an IP (immunoprecipitation) on your sample and then run a western blot.
Hi how are you
I think to get a visible band of your target protein try to load from 25 to 40 ug in each well
then after reacting your gel with your first and second antibodies and detecting your target protein
make stripping of your antibodies and re react again the membrane with antibodies for Beta actin
and then normalize your target protein you visualized before to the newely detected beta actin bands to be able to know your relative protein intenisty
good luck
Hi, I want only to give the advice to load a positive control for your protein expression. You said that your samples are really precious and you have few troubleshoot guide, so I think it is important, after decided the loading amout of you samples, to be sure that your antibody work well.
Good luck
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