IP lysis buffer formulation : why adding a non-ionic detergent if a ? - (Dec/07/2005 )

Dear all,

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 :

QUOTE

One needs to prepare cell lysates if using cells as the source of the protein. Lysis can be done using detergents such as Triton X 100 or by mechanical means such as passing 15-20 times through a 25G needle. If using denaturing ionic detergents such as SDS (usually 1%) or Sarkosyl, remember to add 9 volumes of similar lysis buffer, but with a non-ionic detergents such as Triton or NP-40 (usually 1%), to the lysate before proceeding further. Also, the denaturing lysis buffer should have 5 mM EDTA and 15 U/ml DNAse I.

I'm plannning to use sarkosyl, but why adding a non-ionic detergent ? Is it a kind of "renaturation attempt" ?

Any other (good...) idea ?

-YnB-

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.

-alpha2zee-

Why are you planning on using Sarkosyl?

-Dynein-

Thanks, alpha2zee, for your answer. I suppose that the maximal concentration compatible with IP is antibody-dependent. Any range to suggest ?

QUOTE (Dynein @ Jan 3 2006, 08:59 PM)

Why are you planning on using Sarkosyl?

In my case, it seems to be necessary to disrupt cytoskeletal (myofibrillar) structures. It's very efficient for that purpose

-YnB-

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.

-alpha2zee-