Failure in Immunoprecipitation - No target protein found in eluate (Nov/15/2006 )

Hi

I am perfoming IP to obtain a his-tagged protein from a mixture.

Protocol

5uL anti-his added to 500uL sample (with ~10ug target) to bind overnight

10uL Protein-G agarose added to sample for 2hrs

spin-down and discard flow-through

2 times washes with buffer

50uL SDS-loading dye added to protein-G with 5min boiling

Buffer

250mM Tris-HCl pH 7.5
150mM NaCl
0.5% NP-40
1mM EDTA
0.2mM PMSF

Western Blot Result

Final eluate contains the heavy and light chain of anti-His but not my target protein

Most of my target protein was lost in Flow-through


Is there any problem in my protocol or buffer used?
Any suggestions for me to improve the yield?

Thx Everyone!!!

Hi

I am perfoming IP to obtain a his-tagged protein from a mixture.

Protocol

5uL anti-his added to 500uL sample (with ~10ug target) to bind overnight

10uL Protein-G agarose added to sample for 2hrs

spin-down and discard flow-through

2 times washes with buffer

50uL SDS-loading dye added to protein-G with 5min boiling

Buffer

250mM Tris-HCl pH 7.5
lower ionic strength to ~150 mM, yours is very high; NP-40 with 0.1 % may also do to prevent unspecific binding150mM NaCl
0.5% NP-40
1mM EDTA
0.2mM PMSF

Western Blot Result

Final eluate contains the heavy and light chain of anti-His but not my target protein

Most of my target protein was lost in Flow-through


Is there any problem in my protocol or buffer used?
Any suggestions for me to improve the yield?

Thx Everyone!!!

I never saw such a high amount of tris.
try 10-20 mM
you can also decrease NaCl (or even don't use NaCl), at least for the incubation.

To kosmodrom and Missele,

Thx very much for your advice. I will definitely have a try and reply here my result~~