Beginner Question - (Jul/30/2009 )

Hey guys,

So I'm new to Western Blots, and after having done some ICC to detect for a protein, we now want to do a WB to have more evidence for its existence within a line of mast cells. So my question is, what's the best of way of going about this? I can't just add the protein pellet (solubilized in the loading buffer) because there could be hundred of proteins secreted in this cell, not to mention many around the 16kDa one I'm looking for. Whats the best way of narrowing down the proteins I load, so that I won't get hundreds of bands that end up smearing together? Or is there a way of getting out the much larger proteins out?

Thanks

Uhm... I don't understand... You do have antibodies against your protein of interest, right? So what's the problem? You will have band on WB that has molecular weight and immunoreactivity of your protein - what more do you want?

Answering your question - there are a lot of methods you can apply eg. ultrafiltration, precipitation, chromatography etc.

Eg. ultrafiltration with Amicon centrifuge tubes - first use tube with NMWL of 30, 50 or 100 kDa and collect permeate, then use 3 or 10 kDa NMWL tube to concentrate it.

May I ask what protein expression are you looking for?

Minnie Mouse on Jul 30 2009, 05:39 PM said:

we're looking for leptin

Well, if you are doing a western blot, you will not be seeing every band available. You will be using an antibody that is directed against your protein of interest. Since this antibody (should) only bind to your protein, you can then use a secondary antibody (chemilum. etc..) to visualize it, w/out getting other protein bands, right?

D.Huckab on Aug 6 2009, 01:17 PM said:

Yes, I understand that the antibody yields specificity, but I just thought that if im only able to add about 20ul of cell lysate, and who knows the levels of this protein in the cell lysate, there might not be a significant amount of it relative to all the other proteins that end up getting loaded, to visualize a band.

There are detection limits to westerns, how sensitive they are depends on the antibody. 20ul may be enough, though an actual protein content would be a better measure of how much you are loading (ug? ng? pg?). Initially at least, you may have to do a titration to determine at what level it is best to use your antibody, most should start at about 1:200 and finish at 1:2000.