Preparing supernatant from cell culture for western blot
Posted 27 February 2013 - 08:24 PM
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?
Posted 28 February 2013 - 01:04 AM
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....
I always had an alternate hypothesis....
Posted 28 February 2013 - 05:16 AM
In terms of preparing the sample, you just need to mix the supernatant (concentrated or not) with SDS-loading buffer and load the gel.
Posted 01 March 2013 - 01:12 PM
Posted 01 March 2013 - 01:59 PM
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.