Posted 30 July 2009 - 08:40 AM
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?
Posted 30 July 2009 - 10:05 AM
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.
Posted 04 August 2009 - 05:53 PM
May I ask what protein expression are you looking for?
we're looking for leptin
Posted 06 August 2009 - 01:17 PM
Posted 09 August 2009 - 03:52 PM
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?
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.
Posted 09 August 2009 - 04:29 PM