Extraction of protein from inclusion bodies - (Feb/08/2008 )

Hello guys,

I'm just struggling with western and ELISA these days. I can't see my protein expressed in western blot or ELISA. I would believe they're overexpressed and aggregate in the inclusion bodies. Could someone recommand a buffer to extract protein from that for use in wb or ELISA? They're in plant cellls. Besides, my protein is very susceptible to degradation, is there any good way to prevent the degradation during the extraction process?

You may use culture samples and test for expression of proteins in inclusion bodies by SDS-PAGE gel/Western blot.
I used 1ml (of 1 litre) samples from the culture pre induction, 1 hour-, 2 hours-, 3hours-, 4 hours- and 5 hours post induction and run them on a SDS-PAGE gel to establish the time when expression of my proteins was optimal. Subsequently to lyse samples before loading onto the gel as follows:
Lysis of culture supernatant samples was performed in PBS containing 1% SDS at room temperature for 30 minutes followed by removal of undissolved material by centrifugation at 13,000 rpm prior to running on the gel. An equal volume of protein or lysis samples was added to loading buffer [50 mM Tris-HCl pH 6.8, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, 10% (v/v) glycerol and 100 mM DTT (Sigma)] and denatured at 98˚C for 3 minutes.

However, I must say I do not know anything on plant cells ( I expressed human proteins in bacteria). Hope this helps anyway.

There are several ways to produce proteins susceptible to degradation by use of different expression methods in bacteria (attachment) but I cannot help with that in plant cells.