I made an antiserum in a rabbit immunized with a part of my protein of interest (about 150 KD) fused to Maltose Binding Protein. (The protein fragment alone forms inclusion bodies in bacteria, so it was convenient to work with the soluble MBP-fusion.) Test blots of the crude serum indicate that there is some antibody to my protein, and also a lot of anti-MBP antibody
I then made two affinity resins with 12 mLs of Affigel 15 each: one with about 100 mg of MBP, one with about 70 mg of the fusion protein. About 40 to 50% of each protein prep coupled, based on reduced intensity in dot blots before and after the coupling.
My plan was to purify antibodies on the MBP-fusion protein first, elute them and then run the eluate over the MBP column to remove unwanted anti-MBP and save the flowthrough for staining. I diluted 20 mL of the exsanguination serum in 180 mL 20 mM HEPES-NaOH pH 7.5, centrifuged it 15' 5000g (some pellet formed), and circulated it over the MBP-fusion resin overnight. I then washed with 250 mL each 20 mM HEPES + 0.05% Tween20 than 20 mM HEPES + 500 mM NaCl. I eluted the column with 100 mM glycine-HCl pH 2.5, collecting 3 mL fractions in tubes containing 0.3 mL 1 M Tris-HCl pH 8.3.
Checking the pH of the fractions with pH paper shows they are near neutral. A precipitate formed in the peak fractions soon after the elution. Making dot blots on nitrocellulose before and after a quick spin shows that half to 3/4 of the protein is precipitated. The protein concentration in the peak fraction is not extreme: probably close to 1 mg/ml or a bit more. Any suggestions for an alternate elution method to prevent the precipitation (maybe include salt or glycerol in the glycine buffer?)
Why don't you try to purify the protein first with a protein A column? This will remove probably > 90% of the protein impurities, plus a lot of DNA and other impurities etc.
Then in a second round try your MBP-affinity resin.
Try first to reduce precipitation by lowering the ionic strength of your buffer by half or even a bit more; 30 - 50mM. The combination of low pH and high ionic strength/conductivity is a stress factor for many antibodies.
The eluate coming off the column does not need to have the low pH of 2.5; in optimized protein A chromatography, the antibodies eluate often has a pH 1 or 2 pH-units above the elution buffer pH. It is just important that the protein in the column experiences a low pH for it to elute.
If this still causes precipitation, try eluting at a higher pH, e.g., pH 3.5, which is less stressful for antibodies.
I suggest running the antiserum over only the MBP affinity column to remove MBP reactivity, but not the fusion protein affinity column. This avoids binding the antibody that recognizes the protein of interest, and therefore avoids having to elute it with harsh conditions. As long as the capacity of the MBP column is not exceeded, the result should be an antiserum that reacts with the protein of interest but not MBP.
If you want to purify the total remaining IgG away from other serum proteins, you can use a protein A or G column and elute with a high-salt, near-neutral pH elution buffer instead of a low pH buffer. An alternative to that is to use ion exchange chromatography with a salt gradient elution.
I would only use the affinity column to purify specific IgG to the protein of interest if it is absolutely necessary. The problem with this is that the highest affinity antibodies are very difficult to elute, so you end up depleting them or denaturing them, rather than enriching them.
Upon diluting your sample (200 mL) and loading it onto the column (immobilised with fusion protein) overnight - was this left at room temperature or in a cold room..?
Do you have to load it overnight..?
Not sure whether it will help if you ammonium sulfate precipitate the starting material (serum) with 45-55% AmSO4 (saturation) to remove some impurities and following processing of the pellet (centrifugation and dialysis), filter using a 0.22 or 0.45 micron filter followed by adding free MBP protein to form a complex with the anti-MBP antibody - then filter and load onto your MBP fusion column or used ion exchange chromatography.
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