[biology_06er]

Hi there,

Soo today I eluted with MCAC-100 (or so I thought)..I realsied once i added reducing buffer i added 1M HCL (i have them in the same size bottle next to each other on my shelf)…so what i did was i just washed my column with MCAC-0 a few times and eluted with the correct imidazole concentration …just wondering will this work..im running as gel as i write this but just wondering if the beads are still useable

B_06er

[biology_06er]

or more likely will the HCl remains on the beads affect my protein

[JackBean]

1M HCl has pH ~1, that denatured probably your protein and I’m not sure, whether it doesn’t elute also the ion from column. Which ion are you using? Did you see decoloration of your column?
Do you have protein of which you can measure activity? If so, do it and you will see. You will probably have to do some slow dialysis to re-fold your protein.

[biology_06er]

Hi,

I’m using a nickel column, when i added my reducing buffer to a sample of beads and my eluted protein it turned yellow..so i washed it with my buffer and then tested for colour change…it didn’t turn yellow…so i eluted with 100mM of imadazole and ran a gel and got a band…but i had quite a bit of protein in my flow through so i dialyzed in mcac-0 o/n and tmoorrow i will run it through a new column of beads and elute that correctly..

what do you mean by
Do you have protein of which you can measure activity?

[biology_06er]

Sorry that last message is a jumble of words…so just to explain more clearly..I added 10uL of my "eluted" protein (the one I did with HCl) and added 10uL of reducing buffer-it turned yellow..I also took a sample of beads (10uL and added 10uL reducing buffer–also turned yellow)-..boiled both for 5mins at 95 degrees and then ran a gel…I got a relatively faint band with the HCl elutent but have kept that separetly in the fridge for time being.

After I sonicated, I ran my lysate thru the colum and i ran the FT on gel as well and theres quite a bit of protein there..so I dialyzed in MCAC-0 and tomorrow I will pass it through a new column of beads…and if I have enough there I might just get rid of the HCl one or refold like you say…but yeah I was more curious about what the HCl would do to my protein 🙁

[biology_06er]

sorry to be a pain..if I were to refold it to be on safe side..what to I refold in buffer wise/dialyze..MCAC-0 (Tris 20mM pH 8, 0.5 NaCl–the same as what I washed column with)..this causes the detanurant (Hcl) to be absorbed and hence cause protein to refold?? Have I got that right?

[JackBean]

to re-fold the protein, you should change your buffer/solution very slowly in several steps. Thus, if you had pH 1 and want pH 8.0, I would do at least eight steps, one pH point per step (i.e. first dialysis against buffer with pH 2, second dialysis with pH 3, third with pH 4…).
Does your native protein turn yellow in the reducing buffer. I don’t see much, how is that relevant.

I meant, whether are you able to measure activity of your enzyme or is it some protein, which does not have catalytic activity (at least not something easily detectable in vitro like some transcription factor).

[Author]

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