Help regarding FLAG IP and detergent in elution

Itisam Sarangi via methods%40net.bio.net (by itisam.sarangi from gmail.com)
Sat Oct 4 05:31:35 EST 2008


hi all,
Currently I facing some problems in FLAG IP experiment.
I am using the follwoing lysis buffer glycerol 105, tritonx-100 -.9%
NP40-.1% Nacl 150mM, EDTA.2mM,HEPES 20mM, PMSF .5mM and prot inhibitor 1x,
at ph7.9

I use this washing buffer 150mM nacl, tris 50mM,pH7.4

I perform this expt in a cloumn I have wash 20 column vol of wash buffer.

After that I elute using FLAg peptide. chk the bait protein in silver
stain. Wash and concentrate it through centricon 2 times and send for MS.
yield is in ug level.

But MS is not giving any result becoz of some detergent in elution
(according to people who perform MS for us)

I have tried different wash buffer and centrifuge after each wash cycle so
as to minimise the detergent. and even increase washing more in centricon .
Centrifuge after each wash cycle in my opninion will decrease any
interaction proteins . But at least I am expecting some MS . but still there
is detergent in sample.



I have thought of  follwoing  options :
1) decrease the detrgent to .1% NP40. as some protocols use this

2)  use hypotonic lysis buffer to wash

3)use some other method to remove detergent using kit form pierce or norgen
or beads from sigma



Any help in this matter will be very helpful



-- 
Itisam Sarangi


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