After removing His-tags with enterokinase from mEGFP in pet32b(+) without TEV. I'm measuring protein concentrations with BCA assay and it seems I lost 2/3 of my proteins. I did his-tag removing with enterokinase. After that did affinity chromatography purification with His-trap column. After chromatography concentrated and desalted the protein(mEGFP) with vivaspin 3000 MWCO 20 ml. The changed buffer was phosphate buffer without salts. When doing BCA assays proteins seems to disappear. What could be wrong?
You've done a lot of steps any which could be the a cause of protein loss.
Are you taking aliquots for protein conc. at each step and running SDS-PAGE?
Please confirm your steps:
1: you elute initially from HisTrap to get your mEGFP with tag
2: buffer exchange or dilute to reduce your imidazole concentration
3: add enterokinase and incubate
4: reapply to HisTrap column to capture cleaved tag and uncut material
5: capture Flow Through of cut material
6: elute column for uncut fraction
7: flow through fraction (cleaved mEFGP) applied to Vivaspin to concentrate
8: add phosphate buffer to diafiltrate in Vivaspin?
Run an SDS PAGE of your protein before and during cleavage. If the histidine tag isn't fully cleaved by enterokinase then you are not using enough enterokinase or for a long enough time and so your uncleaved polyhistidine tagged protein is sticking to Ni-NTA and that is how your losing it.
Yeah I did first chromatography to gather mEGFP with tags with His-trap. Then did buffer exchange and concentrate the sample with vivaspin 20 10000 MWCO. The buffer 20 mM Tris buffer without salts. Then add enterokinase to the sample. Did another chromatography were mEFGP was diluted in flowthrough. Get from the fractions his-tags and undiluted sample. Then the flowthrough was concentrate and buffer exchange bt vivaspin 3000 MWCO to 20 mM phosphate buffer without salts. Yeah, I have ran a SDS PAGE and we could see some mEGFP with tags in the gel so not all protein have been cutted but the loss was huge.
Hi, I am purifying His tag protein using Histrap AKTA. But problem is I am eluting protein in all fractions from 4-100% of elution buffer with 500mM imidazole. Protein still come out at 100% elution buffer. So I believe some of the protein is still bound on the column, So, I am not getting protein in peak rather a plateau, a little protein in all fractions. More in impure than pure ones. Then I am pooling out relatively purified fractions, concentrating using 10KD cut-off, doing dialysis and Ulp1 treatment and then running on histrap again. Here I am getting some protein in Flow through and some in early elations (Problem#2). May be some of protein is binding with column even in the absence of His. Later I do concentrate sample using cut off to reduce the volume of buffer and then do dialysis for buffer exchange 4-6 hours to run on Mono Q. The protein I get at the end is quite pure but yield is too little (Problem # 3). I do not know how can I get more purified protein in fewer fractions in Histrap, so I can get better yield at the end
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