I am purifying a protein from a bacterial system and I was able to successfully purify the protein through an affinity column. My protein has a pI of 5.9 and it is stable at higher pH. After affinity I started with IEC using HiTrap Q FF column from GE and my protein is in a buffer of pH 7.5 (50 mM tris, 200mM NaCl, 5% glycerol and 10mM beta ME). However I could not recover my protein after loading the sample in the column. I followed gradient elution from 20-100% of Buffer B (2M NaCl) unfortunately, I could not see any peaks after flow through. I repeated my experiment twice but found no changes in my results (I checked all the samples on an SDS-PAGE). Please let me know if I am going wrong anywhere in the protocol.
Hi Manjula, did you check the flow through to see if your protein was actually binding to the IEC resin?
If your protein is not in the FT and not eluted with 2M NaCL the possibilities are that 1) the protein is still in the column (very unlikely with so much salt being used) or 2) that during the sample preparation the protein has been lost somehow. I assume you filter the sample before chromatography possibly after having adjusted it to the starting buffer conditions. It might be that the change in the conditions in which the sample is prepared before loading it to the second chromatographic step leads to an insolubilisation of the protein than would have then been removed by filtration. Or that your protein is sticking to the filter material, or alternatively there might be a problem in your detection step with SDS-PAGE after the separation.
Hi, if your protein precipitates then it does not matter how much salt you use (unless of course its salt reversible). One could try washing the column with 6M Guanidinium HCl and look at the eluent. If its stuck and precipitated then it should be stripped by this condition. Have you tried running the experiment at different pH values? I don't know anything about your protein but not all proteins are "happy" at that pH. What is the pI of the protein, and, in some cases you may be able to bind to the column even if the "net" charge is not favorable if the appropriate charges form a patch of local charge.
Hi Manjula,
If protein was present at the start point, it must be there in any of the fraction may be in a very low or diluted condition. I think you should try silver staining instead of coomassie, also at the time of running SDS PAGE gel run all the samples like protein before filtration, protein after filtraion(load), flow through, wash and elutions. If there is some specific assay of your protein then try that because assays are more sensitive and works even at a very low concentration of protein. You can also try concentrating your flow through or elutions and then run the gel to get it visible. Alternatively, try TCA precipitation of your samples and then run the gel. Hope this works for you.
GOOD LUCK.
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