Hi everyone,
I have been using MagneHis™ Protein Purification System (V8500) for many years to purify proteins from insect cell culture, Baculovirus expression system. Currently, I have difficulty in purification using this system. I can detect my target protein in the cellular extract by western blot analysis using anti-his antibody, however, I can not detect it or is seen very sligthly, after purification, figure is below. I would be very happy if could suggest me an alternative strategy for protein purification. I would appreciate it very much for your kind help.
Dear Hacer Muratoğlu
2 questions:
You can detect my target protein in the cellular extract by western blot analysis using anti-his antibody, but do you know if this is soluble? Did you check the soluble fraction? Since in case it not soluble is quite normal that you will not obtain nothing at the end of your purification.
Moreover, which kind of detection did you used in WB with anti His? Luminescent detection or colorimetric? Since luminescent WB is really sensitive, it may detect ng or even pg of proteins, but to purify something visible in SDS-page you have to work with ug or mgs so WB is not the best way to assess protein expression since is too sensitive
best
Manuele
Thank you for your answer.
Answer 1: I don't know if protein is soluble. How can I check this?
Answer 2: I used colorimetric detection method for WB with NBT/BCIP substrate.
Can you elaborate;
Cell extract - soluble cell extract fraction or is it whole cell extract?
Also, you don't show a flow through/post binding fraction, which indicate binding occurring.
I suspect that your his-tag is being occluded or has folded back against the protein, if you see little binding.
Depending on your protein, as you appear to have tons, you could use more traditional IEX to do a first pass purification.
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