6 replies to this topic

[wildcatterp]

This is my first time doing a protein purification and I'm pretty stumped at the moment.  I tried eluting my protein from Ni charged NTA beads using an elution buffer first at 250 mM imidazole, 4 degrees overnight in a rack.  After running a SDS-PAGE gel and seeing my protein still in attached to the resin, I increased the imidazole concentration to 1 M first and then to 2.5 M.  I have never successfully eluted a protein before so help would be greatly appreciated!

Marker / empty / supernatant / empty / beads
53 kDa protein
(actual bands are darker)

[jangajarn]

My boss used to say this: whenever you run into problems, go to controls. So, do you have a positive control? like some protein which you know works with this system? May be that the protein is not translated properly. Its been a while since I did protein purification but 2.5M imidazole is not a lot. And, these systems are very senstive to buffer. Make sure your buffer is OK. And, the nature of the protein can also influence elution. Membrane proteins are hard to express, leave alone purification.

[wildcatterp]

jangajarn, on Mar 22 2010, 08:23 PM, said:

My boss used to say this: whenever you run into problems, go to controls. So, do you have a positive control? like some protein which you know works with this system? May be that the protein is not translated properly. Its been a while since I did protein purification but 2.5M imidazole is not a lot. And, these systems are very senstive to buffer. Make sure your buffer is OK. And, the nature of the protein can also influence elution. Membrane proteins are hard to express, leave alone purification.

This is the first time this lab has tried this.  Maybe I will make a different buffer, thanks.  Any suggestions on content other than imidazole?

[wildcatterp]

The elution buffer I have been using is as follows:

100 ul 1M Tris pH 8.0 (final 50mM)

.034 g imidazole (final 250 mM)

.035 g NaCl (final 300 mM)

fill up to 2 mL with H20



Any suggestions?

[vladooo]

purify under denaturing conditions (urea), it works much better than native conditions

[vblanche]

Do you know if your protein is expressed? have you tried an anti-His western blotting on whole cell extracts?

[sumogirlie]

boil the beads in SDS loading buffer, that should at least tell you that the protein is on the beads and can come off