Problems with immunoprecipitation

U5267049 at ucsvc.ucs.unimelb.edu.au U5267049 at ucsvc.ucs.unimelb.edu.au
Mon Apr 11 01:48:26 EST 1994


     Hi everyone,

     I've been having problems with some immunoprecipitation experiments that
I've been attempting recently. Without boring you with all the gory details,
I've been analysing the topological structure of an integral membrane transport
protein in E. coli, by fusing alkaline phosphatase (AP) to varying lengths of
the amino-terminal end of the protein. This produces a series of fusion proteins
of different lengths.

     I want to look at the rate of synthesis and stability of these hybrids
expressed from a low/medium copy vector (6-8 copies per cell). Overexpression of
this membrane protein is deleterious to the cell.

     I had hoped to label the hybrids with [35S]Met and perform pulse-chase
experiments.  I've been following the immunoprecipitation method of Ito et al
(Cell 24:707) as modified by Manoil (Methods in Cell Biol. 34:61). Strains are
grown in minimal medium to mid-log phase, adjusted to the same OD600 reading,
and 0.5 ml of culture is labeled with a mixture of [35S]Met and [35S]Cys
(Expre35S35S from NEN) at 37 C. Protein is precipitated with commercially bought
antibody to AP, and heat-killed S. aureus as a source of Protein A. I've tried
various cell densities in the range of OD600 of 0.5 to 1.0, I've used 10 to 100
microCi [35S]Met/Cys per tube, and labeled for various times between 1 and 5
mins. I've been unable to see any hybrid protein bands even after exposing for
up to 5 days.

     I am confident that the technique and reagents are up to scratch as my
positive control of a strain expressing native AP from the chromosome always
produces a dark band of the correct size. Hybrid proteins are being synthesised
as I can see them in Western Blots. However this technique only indicates the
steady state level of the hybrids, and I need to compare rate of synthesis and
stability.

     Immunoprecipitation of membrane protein-AP hybrids is a common procedure in
the literature, but usually the fusion proteins are expressed from higher copy
number vectors (eg. pBR322). I hope that I am not making some basic error. Any
advice would be greatly appreciated. If this has been discussed in the past I
sincerely apologise.

                                           Joe Sarsero


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