I performed a Thrombin digest of a polyhistidine tagged fusion protein in PBS @ pH7.4 (one of the recommended buffers @ recommended pH for agarose immobilized thrombin) but was not seeing a banding pattern to indicate cleavage (the his-tag should only be about 1kD). To see i have cleaved protein, removed the thrombin by centrifugation and applied the digest (in PBS) to a Talon resin column and collected all of the flow-through. I washed the column with Wash buffer (50mM NaH2PO4 300mM NaCl 20mM Imidazole) and again collected all of the flow-through. I eluted with elution buffer (50mM NaH2PO4 300mM NaCl 250mM Imidazole) again, collecting the flow-through. I have analyzed the flow-through 1, flow-through 2, and eluate on SDS-PAGE. Flow through 1 contains a very small amount of protein, Flow through 2 contains quite a bit, and so does the eluate. I'm inclined to think that this means I did not have good digestion of my protein, however I'm curious to know why there was so much protein in the wash step flow through. Is it because I used PBS rather than a proper binding buffer (which would contain 10mM Imidazole)? Why does the binding buffer contain a small amount of Imidazole?
Phosphate does not interfere with the metal recognition process. Imidazole can and it is added in low concentration to reduce binding of undesired proteins.
I am not sure if the size difference of 1kD for the His-tag is visible during SDS-Page. Alternatively you can do a Western blot using His antibodies. Now, with how many volumes of wash buffer (with 20mM imidazole) did you wash the His trap column? The cleaved protein (without His tag) will bind non-specifically to the resin and you need to wash with 20mM imidazole buffer. If this step is too short you will see cleaved protein in the elution step (with 250mM imidazole). Do you have a Nanodrop machine in the lab? I would check the A280nm (total protein) until you have a very low absorption during the 20mM imidazole wash step (just blank against 20mM imidazole buffer). When the A280nm is down, elute with 250mM imidazole buffer. Analyze via SDS-Page/Western blot.
The concentration of imidazole in the column equilibration buffer, the sample and the wash buffer is critical and may depend on the protein. Too litte imidazole, and the de-tagged protein may bind non specifically to the resin, resulting in poor yield; too much, and the non-detagged protein will elute together with the processed protein. From what you report, it sounds like the de-tagged protein bound to the resin, owing to the absence of imidazole in the PBS buffer used for loading, and that it was then eluted with the wash buffer containing 20 mM imidazole. What came out with 250 mM imidazole is probably unprocessed protein.
If your protein is ≤ 50-60 kDa, you should be able to separate the processed polypeptide from the original tagged protein on SDS-PAGE, even if they differ only by 1 kDa. I would load the unprocessed protein next to the processing mixture and to the separate fraction from the Talon column, taking care of not loading too much sample in order to get good resolution. This way, you should be able to tell what are the species you are dealing with and how they bind to your Talon column.
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