IP lysis buffer formulation : why adding a non-ionic detergent if a ? - (Dec/07/2005 )
I'm totally new in Immunoprecipitation. After a few look on the web, I found in the "Pamela Stanley Lab" cookbook a statement about lysis buffer which I did not understand :
I'm plannning to use sarkosyl, but why adding a non-ionic detergent ? Is it a kind of "renaturation attempt" ?
Any other (good...) idea ?
Sarkosyl is sodium lauroyl sarcosinate, like SDS, is ionic in nature and can be disruptive to many protein-protein interactions, such as those of antibody-antigen or antibody-protein A/G. Thus, it is common to dilute out sarkosyl as well as 'sequester' it with non-ionic detergents.
Note that sarkosyl is not a common component of lysis (protein preparation) buffers. It however is commonly used in such buffers used to disrupt 'tough' items such as nuclei, cytoskeleton, etc.
Why are you planning on using Sarkosyl?
Thanks, alpha2zee, for your answer. I suppose that the maximal concentration compatible with IP is antibody-dependent. Any range to suggest ?
In my case, it seems to be necessary to disrupt cytoskeletal (myofibrillar) structures. It's very efficient for that purpose
The final quenching detergent (such as Tx-100) concentration depends on the final sarkosyl concentration. Note the two final; final is w.r.t the volume of the IP reaction. You can try 1 or 2% Tx-100. I know that 1.5% Tx-100 does not usually negatively affect IP.