lysing mammalian cells in sds - (Apr/01/2012 )

Dear All

I usually lyse my cells in 1%triton buffer, which is not harsh and lets me detect interactions (i.e. co-IP).

However, now I am trying to lyse them (293T or HeLa) in a harsher buffer because I need to make sure all interactions are disrupted (e.g. I want to detect ubiquitination but not binding to ubiquitin). I was told to use 1% SDS, boil, and then correct it using RIPA buffer (e.g. lyse in 100ul 1%SDS and later add 900ul RIPA). I need to correct it because I will make an immunoprecipitation, otherwise proteins wouldn't bind to the antibody. I added some DTT and pH 7.4 Tris-HCl as well with SDS. But once I add this, DNA forms a clump. It seems like proteins are not really solublized. I tried using a syringe, which helps but takes a lot of time and also does not work perfect. Sonification is not really an option as I have small volumes like 100ul. I tried adding benzonase but I am not sure when to add. I tried adding benzonase together with 1%SDS buffer but this does not work.
I would be happy if you have some suggestions.

treatment with DNase I may help

I routinely sonicate 100uL samples with no problems.

Hi

It is not clear what the cell density is when you carry out these operations.

Typically we lyse sub confluent monolayers of cells in 0.5 % SDS w/v in TBS (Tris buffered saline boiling for 5 min). After this you can remove the nucleic acids by ultracentrifugation or by low speed centrifugation through appropriate filters. For a sub-confluent T25 flask monolayers we might use 1 ml of SDS solution (3-4 ml for a T75, etc). The nucleic acid depleted supernatant can be assayed for protein using the BCA assay, using 0.5 % SDS in TBS to make up the standards

I would not recommend sonicating a detergent solution as you may get excessive frothing. Instead, to the nucleic acid depleted supernatant you could add an excess of neutral detergent (e.g. TX-100 or NP-40) to at least 4 times the concentration of SDS. This should be enough to displace the protein bound SDS and allow successful immunoprecipitation.

I hope this helps