Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

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 :

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 ?


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 ?

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 smile.gif


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.