We usually do PEI preciptation to move nucleic acid and ammonium sulfate precipitation before purificating a certain protein,then resolve the precipitate with binding buffer (20mM Tris pH 8.0,300mM NaCl,2.5% glycerol)of histrap with a ration of 1g:10ml, and filter. The conductivity is almost equal to that of 300-500mM NaCl. After that, we load the sample directly to the histrap column,however,the protein can't efficiently bind to the histrap(1ml/min for 1ml histrap).Most protein comes in the flowthrough.I don't think it is overloaded since the eluate is far less than the column's capacity .Is desalting neccessary before the histrap?In my opinion, it is not neccessary. but I am not quite sure.
NaCl concentration is irrelevant for binding of proteins to Ni affinity resin. I would tend to believe that residual polyethylene imine (PEI) is the source of the problem, as it may compete with your protein for binding to the metal (Ni) on the resin. In fact, there is no need to go through the trouble of PEI and ammonium sulfate precipitation prior to loading the sample on the Histrap column. I routinely used to centrifuge the crude lyusate to get rid of debris (20 min 27000 g) and load the crude supernatant directly on the resin. If nucleic acids are a problem (e.g. if dealing with a nucleic acid-binding protein), most of the time they can be eliminated by introducing a washing step with buffer containing 3.5 M NaCl. This does not elute the protein, but it releases nucleic acids that may be bound to it.
NaCl concentration is irrelevant for binding of proteins to Ni affinity resin. I would tend to believe that residual polyethylene imine (PEI) is the source of the problem, as it may compete with your protein for binding to the metal (Ni) on the resin. In fact, there is no need to go through the trouble of PEI and ammonium sulfate precipitation prior to loading the sample on the Histrap column. I routinely used to centrifuge the crude lyusate to get rid of debris (20 min 27000 g) and load the crude supernatant directly on the resin. If nucleic acids are a problem (e.g. if dealing with a nucleic acid-binding protein), most of the time they can be eliminated by introducing a washing step with buffer containing 3.5 M NaCl. This does not elute the protein, but it releases nucleic acids that may be bound to it.
Dear Pierre Béguin，thank you for your suggustion. The proteins with which we deal are usually nucleic acid-binding. An introducing of a washing step with buffer containing 0.5M NaCl, sometimes, is not enough to remove the nucleic acid if PEI precipitation is not done. Will the concentration of the NaCl as you suggested 3.5M be too high? I have never tried.
You could break the nucleic acid into fragments by shear forces (passing through a needle, french press, ultrasonic water bath...), usually that is enough to prevent interference.
Dear Li Lejun
I have used the 3.5 M NaCl washing step many times and it does not affect the retention of the protein to the IMAC resin. However, I cannot vouch that it will not affect your protein, but it is probably worth trying. You can monitor whether it releases nucleic acids by following the A260/A280 ratio in the NaCl wash. In my experience, it is close to 2, meaning that it contains mostly nucleic acids.
Why not adding DNase to cleave the DNA. Is commonly done in protein purification.
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