Re: [ccp4bb] Concentrating purified membrane protein

Raji, One point the most people forget about is that whenever you concentrate any detergent-solubilized membrane protein is that you will ALWAYS concentrate the detergent. So regardless of the MWCO, if the protein-detergent complex concentrates, the overall detergent concentration also increas

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Re: [ccp4bb] Concentrating purified membrane protein

can you elute off the column with low pH ? Or EDTA if you don't want imidazole around ? Jürgen On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote: Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did

Re: [ccp4bb] Concentrating purified membrane protein

Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein.

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Re: [ccp4bb] Concentrating purified membrane protein

Well, you'll never loose the protein in the flowthrough if you don't discard the flowthrough.which i never do until I'm sure about what happened to the protein. But yes, try different Mw cutoff concentrators until you find the highest one that works. Depending on the purification your sample

Re: [ccp4bb] Concentrating purified membrane protein

Hi Raji, DDM has quite a large micelle so you might be alright with a 100kDa cut off concentrator, I've had the same experience with a 30kDa protein in LDAO being comfortably contained by a 100kDa concentrator. I would try with a small amount of you sample and see if significant amounts of prot