I have cloned my insert using Gateway cloning into a his tagged vectors. Then I transformed BL-21 cells once I confirmed my insert. I grew cells with correct selectable marker. I induced the cells using L-arab as per the vector needed. When I tried to purify the protein using ni-nta slurry, I did not get anything. My protein was absolutely not getting expressed. Then I moved on to the yeast system. My new vector has V5 epitope and 6xhis tag. I tried protein expression. So far, I get it in the pellet and it is present in the solubilized portion. But, as soon as I apply the solubilized part to ni-nta, no protein is obtained. I tried it multiple times. I conducted western to confirm my results. I can see my protein till forst wash but it disappears after that. My washing solution does not have immidazole. I really do not understand the problem. Please can anyone help?
P.s: my protein is 40kDa
Quick question for the western did you use a His antibody or a specific antibody for your protein? I ask this to make sure your gene is properly cloned into the vector and the His tag is expressed along with your gene. I had not problem when using the pET28a system with IPTG.
I probed it with anti-v5 antibody. I had sequenced my vector to check if everything is in frame. But, I do not know a way to check whether my his tag is expressing or not.
It will be good to obtain a His antibody, although if everything is in frame I don't see why His tag wouldn't be expressed. Since you tried this with two different systems and the common step is the ni-nta purification, the problem might be there. I wonder if your ni-nta agarose is too old or not stored properly and that is what causing the no binding. If you have another construct that you know has worked before, you might wanna use it as a control to test your ni-nta.
I used tris buffer. Should that cause a problem with nickel slurry?
Buffers with secondary or tertiary amines will reduce nickel ions, but I have used 50 mM Tris or HEPES, pH 8.0 (check the pH of your buffer as well) for purification on nickel. Avoid EDTA or EGTA in your lysis buffer, check if you are using a protease inhibitor cocktain that it doesn't have EDTA or EGTA in it - this will strip the nickel from your column and would also effect binding.
Sodium phosphate or phosphate-citrate buffer are the recommended buffers for use with Nickel columns. Check the pH of all of your buffers - especially your wash buffer to make sure it's not too low.
What Kim suggests is a good point, pH is critical. The buffers I use for denaturing conditions is 100 mM NaH2PO4, 10 mM Tris, 8 M Urea (pH 8.0) and for native conditions 10 mM Tris, 10% glycerol, 0.5 M NaCl, 0.1 % NP40, 5 mM DTT (pH 7.9).
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