3 replies to this topic

[cellgene]

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.

[Inmost sun]

treatment with DNase I may help

[doxorubicin]

I routinely sonicate 100uL samples with no problems.

[alan.hargreaves@ntu.ac.uk]

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 [pH 7.4] 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