A forum to address questions regarding methods of protein purification. | Contact experts in Protein Purification to get answers
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Questions related to Protein Purification
I am planning on purifying a small 12 kDA protein with a 6x-His tag using Ni-NTA resin. Many protocols recommand the addition of protease inhibitors like PMSF for optimal lysis and purification of...
09 September 2019 594 5 View
Do we need to place the insert in a specific site in his tagg vector for further protein purification or can we just use any restriction site present in the vector?
09 September 2019 8,375 3 View
I need IPTG to induce big volumes of E.coli cultures to do protein purification. Many suppliers advertise their IPTG as being "dioxane-free". What difference does it make ?
09 September 2019 6,644 4 View
We have a cellulase crude protein mixture with oligosaccharide contamination. I have tried various protein purification methods like Ammonium sulfate precipitation, Column based, TCA method,...
09 September 2019 8,455 7 View
Hi everyone, The DE-52 column is equilibrated with [pH 8.0 20 mM Tris-HCl, 1 mM EDTA] buffer, and the pH is around 8.5 at 4 deg (checked with pH meter). The Mb (pI ~ 7) is dialysed overnight...
09 September 2019 7,699 10 View
I'm attempting to concentrate whole cell lysates from primary murine cells, in order to load enough protein onto a gel to detect my protein of interest. I'm pretty new to protein purification and...
09 September 2019 7,629 3 View
hi, i'm doing a protein purification and i'm wondering what are the factors make a single SEC peak give many bands in a BN-PAGE.
09 September 2019 7,668 8 View
Hello everyone, I try to purify a chitin binding domain in a native conformation in mono disperse suspension. After purification by use of a polyhistidine tag I dialyze the protein suspension in...
14 August 2019 2,980 6 View
Hello all, I am currently reading up on clear coli BL21(DE3) cells as a possible alternative to BL21(DE3) cells for recombinant protein production such that my end products are not contaminated by...
08 August 2019 2,996 3 View
08 August 2019 2,852 7 View
I am trying to concentrate enveloped RNA viruses from infected cell culture supernatant. The clarified media (MEM containing 0.375% BSA fraction V.) will be mixed with PEG8000( to a final...
03 August 2019 437 3 View
I'm trying to express recombinant protein from mammalian cell 293F by its secretion in to the supernatant and then using the Ni-beads to enrich the His-tag protein. However, when I use 300mM...
11 July 2019 4,699 5 View
I have seen lots of papers in literature using prep cell for purifying nucleosomes (from DNA and hexasome species), but I couldn't seem to find another protocol apart from that discussed by Luger...
07 July 2019 6,861 4 View
07 July 2019 2,157 6 View
Is there a more stringent elution buffer than 2% SDS with BME? I have tried this both at RT and 50 degrees with very little antibody eluted on SDS-Page. Based on ELISA, the antibodies are...
07 July 2019 3,042 4 View
I am attempting to purify a protein with his-tag. Current lysis method includes his-lysis buffer (pH8) with 1% triton and 2minute sonication (pulse 10seconds, rest 40 seconds) after incubation...
07 July 2019 3,030 10 View
I have a set of buffers (Tris, imidazole, phosphate) for NI-NTA protein purification. I prepared them about three weeks ago and they contain DTT and PMSF. Can I add fresh DTT and PMSF to the...
07 July 2019 3,503 4 View
I have purified a protein, however I am getting double band on SDS-PAGE. Even after FPLC the double band doesn't resolve and become more prominent. I have checked through Mass spec that both the...
07 July 2019 6,798 10 View
06 July 2019 370 3 View
04 July 2019 2,706 3 View
We just bought the new strep-tactin XT for protein purification. The manufacture recommends to use 50 mM biotin to elute the protein. While 2.5 mM desthiobiotin is suggested for protein...
14 June 2019 5,513 5 View
We are planning to extract RNA from mice tumors. Our downstream application will involve doing quantitative PCR (from cDNA). Does anyone have experience what is the best protocol of RNA isolation...
10 June 2019 2,235 6 View
06 June 2019 5,010 4 View
protein purification, protein concentration
06 June 2019 6,200 6 View