His tag protein purification problem - (Jun/12/2004 )

Hi guys,
Sorry to interrupt. I have a question and instead of starting a new thread I thought I'd ask in here as its kinda related.
I too am trying to purify a protein (containing histag). I am tryin to pulldown the protein with resin treated with Nickle.
It doesn't seem to be working.

My procedure:
- grow protein
- keep soupernatant (throw out cells)
- Add Nickle treated beads to soup, rock for 30-45min
- Spin, wash with 1M NaCl & 20mM Tris (x2)
- Add beads to epi, take out excess wash and ad 50ul LSB.

Western results aren't that great.

Any tips?
Should I just do column purification instead?

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Try playing with Buffer,
Phosphate instead of Tris, and also the pH should be 7 - 8 or more.

Lower pH is usually used for elution from Ni NTA apart from imidazole.

Keep the salt constant in binding and elution.

If it works with Pull down(batch method) it will work in column, if it doesnt work there it wont work in column as well.

have a great day,

Sankar

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all

I have already registered to this forum. I have a problem about His Tag restriction from my 40kD protein. I am trying to optimization of DAPase and Qcyclase restriction but I have no positive result so far and I could not find where I am wrong. If you write any comment or your experience I will be very pleased

Thank you before hand

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Another possibilty is spontaneous loss of the His tag, which you can detect by mass spec. We have observed this in quite a few of our recombinant proteins (that don't contain protease cleavage sites). I strongly recommend that you submit all of your recombinant proteins (especially mutants) for mass spec analysis (e.g. electrospray)

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Hi every one,

I am working on a ATPase protein which is HIS-tagged. I go like, lysing in 20mM Tris and 500mM NaCL, and elute at 250mM Imidazole. But once after this step the protein is very pure and its getting completely aggregated. I tried adding b-ME sill no difference.

I am just wondering that is thr any chances like any other substances I can add to stabilize the proein.
I appreciate your help.

Thank you.

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I have been working on purifying a protein that is 28kDa in size. I have read literature on purifying the same protein, some papers suggest denaturing the protien and other say to use native conditions. I have tried both, but have never had the same recovery as described. Here are my methods:

I do an O/N culture of the bacteria, then the following day I grow the bacteria in steps, first 10 ml to OD 0.3, then 100 ml to OD 0.3, then 500 ml to OD 0.3 then I induce 500 ml.

I have been using B-PER (Pierce) to lyse the bacteria, I also add DNase, lysozyme and protease inhibitors (without EDTA). I spin the lysate at 10,000 rpm for 30 minuts, but there is very little insoluble material.

I then proceed to the batch method. I use the Cellthru cobalt resin for purification. I add 1 ml of resin to 5 ml of lyaste and 44 ml of Native buffer, which has 50mM sodium phosphate pH8.0, 10mM Tris-HCl pH 8.0, 450mM NaCl, and 10mM Imidazole. I let this rock at 4C for 1 hour, then I wash the resin with the same native buffer. After I wash, I elute with a solution that has 50 mM sodium phospahte, 20mM PIPES, 450mM NaCl, 250mM Imidazole, and 100mM EDTA.

I know the EDTA strips the column, but if the protein is pure, then I just need to get rid of the EDTA and I am sure I got all the protein off.

My only problem has been that I loose a large amount of protein in the flow-thru. I have been freezing the flow-thru and using it again and I am able to recover more protein. Is there a way I can maximize the amount of protein that binds to the beads so I can elute all the protein from the column. I am trying to recover high volumes of protein, but it typically takes me weeks to recover enough protein. I need to get 10mg/ml of protein for the experiments I run. Does anyone have any suggestions?

Thanks!

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sankar,

I would try to use Guanidine instead of urea. the washes are made with urea. only the cell pellet is solubilized in guanidine and sonicated previously before aplication into the resin. take a look to the protocol of probond from invitrogen. I had similar problems and this worked for me. Just use TCA to pp your protein before sds page.

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I am using the Qiagen expression system and have had similar troubles. I bind my denatured protein extract to His beads in 8M urea but the protein never elutes. I took some beads and boiled them to see if the protein was there and it was still bound in large quantities. I have eluted with 500mM imidazole and 250mM EDTA. There are still many proteins bound to the beads. What do you think this is a result of? I am using 6L prep which had very high expression. Could the expression be too high? Is it clogging the column? Also, we used sonication to lyse the cells. Could this have caused DNA to clog the column? I bind the extract in a batch manner, but elute with a Biorad plastic column at room temp. Any suggestions are helpful.

Jon

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You may require higher imidazole concentration to elute your protein. We have used upto 1 M in cases. If you find large amounts bound to the beads it maybe that your protein shows very high affinity. Do you know whether the protein has his- or cys-patches that may increase affinity for His beads? If you think you have problems with DNA you should try benzonase treatment. It is very effective.

José

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It sounds to me that you have some sort of secondary structure in your his tag, or that the his tag is causing ppt to occur. The fact that you can bind it to the nickel column when it's unfolded is promising, and, in fact, there's a whole section in the Qiagen booklet about refolding proteins on the column. Perhaps this is something you can try!

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