May 20, 2012 at 3:43 pm #16512LittleBirdParticipant
the question may seem dumb at first sight, I hope all the same it’s not too stupid to ask….
It can be reduced to "why is it – in this case – necessary to perform a dialysis?"
We transfected HEK293T in order to express a his-tagged protein. The cells were kept in DMEM (high glucose, modified with 5% FCS and 1% streptomycin/penicillin), which was collected daily and exchanged with fresh modified DMEM.
The collected supernatants where then dialysed against a his-dialysis-buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8). Afterwards Ni-NTA Agarose was added and finally these beads where put on a column.
Some washing procedures followed (his-dialysis-buffer with either 10 mM imidazol or 50 mM imidazol) and in the end eluted (250 mM imidazol).
I know that a dialysis is in general necessary if buffers need to be changed, one wants to get rid of some certain ingredients/contaminations/salts etc. but somehow I don’t get why it is necessary here – what is in DMEM that would make it difficult (impossible?) to run the ni-affinity chromatography? Would the protein precipitate? If yes, why then?
I’ve been using google for the past days with different combinations of keywords and somehow don’t really get the point – as mentioned above – I hope it’s not too stupid to be asked.
Thanks in advance, every hint and help is appreciated.
May 20, 2012 at 5:41 pm #111168
The protein was expressed extra- or intracellulary? What’s in the medium?
May 20, 2012 at 5:55 pm #111169LittleBirdParticipant
as far as I know expressed intracellulary and then secreted into the medium.
The medium is casual DMEM (http://de-de.invitrogen.com/site/de/de/ … n.170.html), with high glucose and L-Glutamin with 5% fetal calf serum and 5% penicillin/streptomycin.
Or do you mean anything else?
May 20, 2012 at 8:16 pm #111170
Of course the protein is expressed in the cell. But it is secreted. I guess you have removed the cells and probably concentrated the medium and exchanged with some buffer, right?
The medium contains some histidine, but at low concentration (0.2 mM). More important will be probably pH of the medium, because it is not buffered.
July 22, 2013 at 12:28 pm #114112sullivanParticipant
I attempted to purge my protein from the unit society supernatant. I transfected my units with my develop to prepare my protein in the supernatant. I gathered the supernatant around the range of 1 litre and after dialysis I subjected my supernatant to cleaning yet I don’t get my proteins at the closure. Do you have any suggestions?
July 29, 2013 at 10:51 am #114126
did you check somehow where you lost your protein or whether it was expressed at all? How do you detect your protein? Activity, Western?
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