Hi, if your his tag is shielded then your protein will not bind to the column, but your protein does elute from the column which rules out the possibility of problem with his tag ( thats because when you are using specific monoclonal abs against eluted fractions you are able to detect bright band of your protein). So his tag is working means you have to change your his tag antibody or try from other supplier or try different concentration and find out which concentration is best for you. You can use Dnase Rnase during lysis.
Hi Lindsay
Is better if you post images from the gels and chromatograms for our understanding (Redacting confidential parts, etc...if needed).
One question, did you get exactly the same results in samples coming from e.coli and from Sf9?? (I'm supossing taht you're secreting the protein to the broth in this last one)
If the expression of the protein in soluble form is poor, then you will not get it pure using just a Ni affinity column. If the protein is aggregated but soluble, it will probably not bind very well to the Ni affinity column, but what you elute will show up at high molecular weight on the gel filtration column. My guess is that your protein is at least partially aggregated, and there is not that much in the soluble fraction.
Ramon Roman Roldan I added pictures of the elution profile, with SDS PAGE and the final protein with both SDS PAGE and Western.
I get the same purification and Western blot results with Sf9 and EColi, but the elution profile on SEC is not the same. The protein is intracellular in both cases
Adam B Shapiro I am getting pure protein from the Ni column and the aggregation according to the A320/280 ratio is very low. The strange thing is that the protein does not work on a western blot with and anti-His antibody, even after trying different dilutions and different antibodies.
Also, I am very curious about the first peak on my SEC run, where the A260 is double the A280. I was not expecting to have any DNA or RNA in my sample. The protein is a nuclear protein, but it is an enzyme that catalyzes citrullination. It is not a transcription factor or an RNA binding protein.
The severe tailing of the gel filtration peaks is troubling. There might be a problem with your column.
Well, i'll not be worried about the AntiHis antibody failing to detect Histag. HisTag is here and working, or you won't be getting any protein in your IMAC elution. AntiHis antibodies are sometimes unreliable due to the small epitope and the possibility of proteolysis of the Histag during sample boiling. Perhaps you could try lowering the boil temperatures, but as long as you're detecting your protein using an specific antibodie, i think that won't worth the effort.
On the other hand, your SEC profiles are quite informative: If i understand lane labels, you're getting you're protein in two separated peaks: One have more or less the expected mw and spectre (As Adam B Shapiro pointed out, with this tailing is seems form me that the mw determination with your SEC colum may not be totally exact) and other that is way bigger and with typical DNA spectrum.
So, it's seems that you're co-purifying with DNA in your IMAC, althoug you dind't expect (It's happended to me sometimes, it's not so weird), probably with other DNA binding proteins, wich explain the lower purity. In order to confirm, run the big SEC peak in agarose gel , Bioanalyzer or similar. If you find DNA, then you have it, you'll need to treat culture broth with DNASe or precipitate DNA before purification (You'll need to optimize to not affect protein yield.)
If your protein that was expressed in E.coli might contain more contaminants. One possible reason could be the lower induction temperature. You may induce your protein with relatively higher temperature and shorter time. Also i think you can use DNAase RNAase during lysis. Also you may need to optimize your buffer condition according to the pI of the protein
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