I am purifying my his-tagged protein with GE His-trap 1ml column. I started with equilibration of the column with 10mM and 20mM imidazole, and washing has been raised to 50mM, 100mM, and 110mM imidazole with elution at 250mM. The problem I encounter is that I have little protein elution and there is an extra protein elution at 250mM with my protein. Please suggest some conditions.
Hi, I have a question? Do you elute at 250mM or do you do a gradient? If you are sure your protein is binding to the column and it is not eluting on the washes what I would do is that I would try with an Imidazole gradient, I use 0 to 500mM and then identify on which fraction is my protein on an SDS gel. That way I can separate my protein from contaminants and if necessary I do an extra purification step on a Source15Q column
Do what Alexa said. Also what is your salt concentration in the buffer?
I have found with several His-tagged proteins that the best way to bind the protein to the media is in batch mode for >1 hr at 5oC with gentle rocking, then pour the resin into a column.
I am using 300mM NaCl and 50mM NaH2PO4, I am using imidazole as 10mM, 50mM, 90mM , 100mM and 110 mM, there is very little elution of my protein at 110mM, and i elute at 250mM, at which there is some little contaminant, which retain at first 2ml elution and then disappears. in last 3ml. Could it be that if i increase my imidazole to 500mM, there will be more elution product. Because I have very little elution.
Hi Muhammad, I think the best approach would be (or at least that is what I would do) to bind the protein doing a batch (like Gary said) if your column is not pre-packed if it is prepacked then you can do a "batch" using a pump so you recirculate your protein over and over and ensure the maximum protein gets bind. Then you wash with Imidazole, I use 30 or 50mM and then do a gradient (0-500mM). The advantage of doing an Imidazole gradient is that you use a wide range of imidazole and can see aprox on which Imidazole concentration your protein elutes the best. I think that if you use a fixed Imidazole [ ] you are kind of guessing.
Muhammad,
It sounds like you think the protein is still bound to the column after the 250 mM imidazole wash. Have you taken ~10-20 uL of the resin "from that column" and heated it in SDS/Loading buffer at 95oC for 5 min. and run that on a gel to see if the protein is still bound to the resin? The columns can be cracked open fairly easily by using a box cutter razor blade to "carefully" cut a groove just under the top red cap of the column, and then while holding the body of the column in your fingers force the cap against a hard surface to break it off. In my experience, 250 mM imidazole will elute all of a His-tagged protein from the column.
@ Alex and Gary, I have used resins that did not worked for binding, i am using prepacked column from GE 1ml, I have used with syringe and AKTA. I used 2ml to purufy but the binding was extremely less. most of my protein was pressent in the flowthrough. So how i can improve this binding.
Okay, then you need try batch binding "without" any imidazole in the lysate buffer: mix 50 uL of resin with 1-5 mL of protein lysate and incubate for 1-2 hrs at 5oC with gentle rocking to keep the resin suspended. Then gently centrifuge the lysate/resin and take 10-20 uL of the resin (cut the tip off a P200 tip to make it larger so you can pull up the resin) and mix with SDS/PAGE buffer and heat for 5 min. at 95oC and run on a gel to see if your protein bound. Once you get good binding you can try adding imidazole to the lysate buffer to reduce background binding, then scale up to 1-2 mL of resin for ~50 mL of protein lysate (depends on protein level) and pour resin/lysate into a HR column.
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