SDS PAGE Inclusion bodies - (Feb/13/2008 )
Hi,
we have our recombinant human protein expressed in incusion boies in E.coli. we would like to determine the level of expression at different time intervals by performing SDS PAGE of Inclusion bodies. but this IBs shall be contamianted with cell extract so can anyone provide me a protocol to perform SDS PAGE of Incusion bodies.
Thanks in anticipation
we have our recombinant human protein expressed in incusion boies in E.coli. we would like to determine the level of expression at different time intervals by performing SDS PAGE of Inclusion bodies. but this IBs shall be contamianted with cell extract so can anyone provide me a protocol to perform SDS PAGE of Incusion bodies.
Thanks in anticipation
You may use culture samples and test for expression of proteins in inclusion bodies by SDS-PAGE gel comparing the abundance of the band with the size of interest before and after induction of protein expression: 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.
You can 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 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.
I used BL21(DE3) pLysS to express proteins (from pET3d vector) by induction with IPTG. In this system the optimal harvest of the proteins to be expressed was usually achieved after 4 hours post IPTG induction (first band visible post 2 hours).
Any help?
Good luck!
Thanks very much for your reply. i will certainly try method described by you.
however, i needed few explanation. what does culture supernatant sample mean. Is it the same as culture sample or do we have to centrifuge the culture and take supernatant or pellet. At what ratio do i use culture and PBS. is washing with water sufficient to remove the unwanted material and how much washing and at what ratio will be good enough.
sorry for the bothering.
thanks