With primary monocytes/macrophages, we are getting a dirty signal with multiple bands and background using otherwise specific antibodies that usually give us one band.
We recognize that flow cytometry is usually the go-to to look at these sorts of populations, but for this particular experiment, flow cytometry is significantly more difficult than western blot.
Hello A.M.V
How do you prepare your samples? Do you denature proteins right away? I had problems with my cells before I included protease inhibitors when trying to isolate native extract. Proteins will anyway be denatured if you use SDS-PAGE before blotting.
Please include more information about your protocoll to allow for more precise answers.
Yours, IW
We do standard lysate preparation with SDS-containing lysis buffer and addition of Laemmli sample buffer for western blot. We have limited experience with primary human monocytes/macrophages, so we aren't positive whether there's another step we should be including. The literature also varies, it seems.
With primary human macrophages, we use RIPA buffer or, preferentially, T8 buffer (Urea 7 M, Thiourea 2 M, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) 3 % (w/v), dithiothreitol (DTT) 20 mM, tris(2-carboxyethyl)phosphine (TCEP) 5 mM) with P8340 protease inhibitor cocktail (http://www.sigmaaldrich.com/catalog/product/sigma/p8340) to lyse the cells and then we add SDS-loading buffer prior to running the samples on a PA gel.
What type of samples do you have? If it is full blood than the so-called background bands can be Immunoglobulins. I have seen this before.
What is your gel and blotting protocol? How do you block etc?
We do positive selection for monocytes from human PBMCs and stimulate with different interleukins, culturing on standard 6-well plastic plates. After the sample prep steps that I mentioned above, we have been running 30ug on a standard SDS-PAGE gel and blocking in 5% milk in primary overnight. The proteins we are looking at we know are expressed by macrophages, we just need to get rid of all the excess bands.
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