Preparing supernatant from cell culture for western blot - (Feb/27/2013 )
I'm trying to measure a secreted protein. Will the protein lysate preparation be similar to how you prepare cell lysates?
Do I need still need to shear DNA in supernatant samples? (I presume no, since I'm working with adherent cells).
What housekeeping protein (tubulin, actin, GAPDH etc) will you use with supernatants?
I personally not experienced but just an info i know, usually people concentrate the supernatant to a very lower volume and then proceed western as usual, i dnt think u ll have to shear DNA, even if it suspension cells as you can filter the medium and use.
there are some manual techniques that are used to concentrate, but i have seen using centrifugal columns tat comes with molecular weight cut off, meaning depends on the molecular weight of protein we want to analyse, can use the appropriate column to filter all the proteins around or upto that molecular weght from any biological solutions. later you can directly denature them and load.
may be experts here may give you some more inputs and reg the specificity control....
One thing to take into consideration when running supernatants, is the presence of serum in your media. This will come up as a big blob that can give pretty bad background (I believe due to the IgG present in the serum) and can sometimes mask your protein of interest. I think it runs in the 50-75kDa region, but not 100% sure about this. Concentrating the supernatant will concentrate the serum. The serum will also mask any protein concentration measurement you might do (in case you were thinking of doing this for normalisation).
In terms of preparing the sample, you just need to mix the supernatant (concentrated or not) with SDS-loading buffer and load the gel.
The serum protein you'll see is albumin (~60kD) and there is a lot of it in cell culture supernatant unless you didn't add FBS or FCS to your cultures. You'll often see a white 'ghost' blob on your western ~60kD if too much albumin was in your sample. Bovine IgG is also present if not using a low bIgG FBS but not to sufficient quantities to mask things on your blot unless your protein of interest is ~150kD. If there are antibodies available to your protein and a source of purified protein, you may want to consider running a quantitative ELISA instead - then serum componenets won't really matter.
Missle on Fri Mar 1 21:12:49 2013 said:
The serum protein you'll see is albumin (~60kD) and there is a lot of it in cell culture supernatant unless you didn't add FBS or FCS to your cultures. You'll often see a white 'ghost' blob on your western ~60kD if too much albumin was in your sample. Bovine IgG is also present if not using a low bIgG FBS but not to sufficient quantities to mask things on your blot unless your protein of interest is ~150kD. If there are antibodies available to your protein and a source of purified protein, you may want to consider running a quantitative ELISA instead - then serum componenets won't really matter.
Or you can collect your supernatant of interest in a serum-free media or serum replacement media.