Hi everyone,

We are purifying a protein from E. coli and need to get rid of RNase contamination remaining at the end of the purification.  The last purification step is gel filtration, which should get rid of any RNases (I thought), but our user tested for RNase activity and told us it is still present.

Are there any other tricks for removing RNase contamination?   Someone told me to increase the salt to very high concentrations in the lysis buffer.  We thought EDTA might help, but we don't want to add EDTA as the protein also binds metals.

Thanks for any advice!

best,

Peggy

Hi Peggy,

I have just uploaded a protocol (in the Files section) that I had prepared a couple of days ago for someone who had the same problem with removing RNase activity. Ideally your protein passes through an anion exchange resin without binding which removes most of the RNase activity. Then you need to repeat the high-salt washing steps at least twice, more likely 3-4 times until you can't detect any significant RNase activity.

Note that it might be necessary to clean your purification system completely after each run with 6M Gua to avoid any risk of running your purified sample over a "contaminated" system.

I hope that helps.

Best regards

Hüseyin

I never try Husseyn suggestion; they sounds very good.

I would take in consideration that you can later contaminate your samples with additional RNases since they could be in every place: fingers, buffers, etc. If the protein that you need is extremely sensitive to the presence of RNases, I would suggest to steilize your buffers in the autoclave (I remember that once we have a glycerol bottle with huge RNase contamination), wash all your AKTA system and columns with 0.5M NaOH and use only buffers that were previously autoclavated. Use new tubes, perhaps there are RNase free tubes (I do not know)

Mario

Hi Hueseyin and Mario!  Thanks for your suggestions!  I am not sure where the RNAse comes from, I suspect it is a combination of some remaining during purification and some activity in buffers, etc., as Mario mentions.  We will clean our FPLC and column and try Hueseyin's purification strategy.  Thanks again!

best,

peggy

Hi Mario,

just a short comment on autoclaving the buffers. Unfortunately the RNase activity has been shown to survive autoclaving due to refolding of denatured RNase. In order to minimize the RNase activity DEPC-treated buffers should be used (1ml DEPC in 1L, incubate overnight, then autoclave).

Hüseyin

thanks Husseyin; I did not know about DEPC. It looks fantastic. During my PhD thesis I worked with a specific RNase from picornaviruses, and it was terrible to eliminate or at least believe that you eliminate or other RNase. It was before DEPC. It shows how old I am.....By that time I only worked with fresh autoclaved buffers...

Hi again,

Just a note about DEPC - I think it is not compatible with phosphate buffers.

best,

Peggy