I purify my protein of size 65 kD by affinity Chromatography using Ni-Charged chelating sepharose, followed by Gel filteration Chromatography using Superdex 200 HB 10/30 column (Amersham, Piscataway, USA). Although, i achieved purity, however i do not get a single band of my desired protein as shown in SDS-PAGE image [Lane1 Marker 2.Affinity Purified Protein.3-10 Gel Filteration Purified Protein.]
you are mainly getting chaperones at 70kda and 30kda. ..usual problem.... they dont hinder the experiments much but cant be sure. u can try to wash the resin with high salt (1M NaCl/KCl) if ur protein is salt stable and imidazole (20-30mM). rest of the bands are degradation of your protein...use 10% glycerol throughout ur buffers....perform all the steps in cold...induce ur protein at low temp (15C) for 10-14hrs...decrease the time for binding with resin (1 Hr max). anyways u shud get gud protein if u run acta (gel filteration) after affinity purification and concentrate it further. hope this will help.
@Andaleeb Sajid. Thanks dear for your nice suggestions. Actually i purify acetolactate synthase [biosynthetic enzyme]. i used higher imidazole upto 500mM during elution step. Further, i do concentrate the protein prior loading Gel filteration coloumn. Besides, i carry out all the steps 0- 4 degree celsius. As you suggested, i would decrease the binding time, probably this would avoid binding of undesirable proteins. But could u please let me know, the effect of 10 % glycerol throughout buffers.?
Without knowing the exact experimental conditions and seeing the protein extract of the control (untransformed) host cells I am not convinced that you even need gel filtration at all. The nickel binding is usually much stronger than unspecific protein-resin or protein-nickel interactions. So unless the undesired lanes are degradation products with the His-tag, you should get pure band if you wash the column with JUST a bit less imidazole that would elute your protein. I would use an imidazole gradient and getting fractions for establishing the exact concentration where your protein starts eluting. Washing the column extensively with appropriately chosen concentration you should get rid of all the undesired bands on the expense of loosing a small fraction of the protein. High salt or other tricks can also be useful for braking possible protein-protein interactions of proteins belonging to complexes, to prevent co-purification, in general.
increase washing time that will give u purified single band.in my case i increased wash step got more pure protein.
Test the purity by purifying with different concentration of imidazole earlier in broad terms and then narrowing the gradient
10% glycerol works as antifreeze...helps protein to remain intact without degradation during freezing/thaw from -80C. during purification at low temperatures also needs glycerol....one more important troubleshooting is...slow down the binding and washing...decrease the flowrates....really helpful for good quality...
hallo, may be you can use Acid Urea Polyacrlamide gel electrophoresis instead from SDS-electrophoresis. may be with AU-PAGE yor can see a single band.
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