Protein purification issues - (Sep/14/2008 )
Hello I'm a student working in a lab and I stumbled upon this forum and would obviously like some form of assistance with my dilemna.
I was purifying a whole set of proteins that were supposed to catalyze a certain rxn and I later found out I was supposed to remove glycerol from my washing and elution buffer because it is a substrate for the enzymes I am purifying and I need to do functional analysis later so I obviously can't have glycerol in my purified protein solution.
Anyways, I am performing affinity chromatography using His-tagged proteins and Nickel.
I was told to remove the glycerol from my washing and elution buffers so I did that.
So I simply removed the glycerol and replaced it with an equal volume of water.
Now I can't seem to get any protein.
These are proteins I alrdy purified and got b/w 10-30mg/ml but now I'm lucky if I get 1mg/ml.
The odd thing is I just got 1 protein at 10mg/ml yesterday.
However, there must be something wrong here because my results with the purification should be reproducible to some extent.
I remade my washing and elution buffers just incase I made a mistake. I can't think of any reason why this shouldn't work.
I'm also on a fairly tight schedule and my PI seems rather unimpressed with my progress.
I was purifying a whole set of proteins that were supposed to catalyze a certain rxn and I later found out I was supposed to remove glycerol from my washing and elution buffer because it is a substrate for the enzymes I am purifying and I need to do functional analysis later so I obviously can't have glycerol in my purified protein solution.
Anyways, I am performing affinity chromatography using His-tagged proteins and Nickel.
I was told to remove the glycerol from my washing and elution buffers so I did that.
So I simply removed the glycerol and replaced it with an equal volume of water.
Now I can't seem to get any protein.
These are proteins I alrdy purified and got b/w 10-30mg/ml but now I'm lucky if I get 1mg/ml.
The odd thing is I just got 1 protein at 10mg/ml yesterday.
However, there must be something wrong here because my results with the purification should be reproducible to some extent.
I remade my washing and elution buffers just incase I made a mistake. I can't think of any reason why this shouldn't work.
I'm also on a fairly tight schedule and my PI seems rather unimpressed with my progress.
Maybe it has to do with when I add IPTG to the bacterial cultures and how long i leave the cultures to shake for. I usually add IPTG at 0.7-1.2 OD600 and leave it overnight (4pm to 12pm next day) at 16degrees Celcius in the shaker.
What's the rest of your buffer? If you just use water to load onto the column, it's not really surprising the proteins aren't happy.
Do you know why you had glycerol in the buffer in the first place? Find that out, and you should be able to decide on what to replace it with, if anything (as a starter, I would probably try just making the lysis buffer without any glycerol, loading and eluting as normal. You'll probably find the prep isn't as clean; if so, look at changing the salt concentration, to see if you can reduce the non-specific binding).
The washing buffer and elution buffers contain imidazole, NaCl, and Hepes/Tris at 7.5pH. Ill have the concentrations and volumes as soon as possible but I'm not at the lab at the moment.
My PI continues to suggest that the glycerol isn't too important. I'll fgure what it was used for soon enough. Ya...i know. It's stupid that I don't know why it's in there.
And I no longer think the cause is IPTG induction.
I don't use a lysis buffer---sonication.
My PI continues to suggest that the glycerol isn't too important. I'll fgure what it was used for soon enough. Ya...i know. It's stupid that I don't know why it's in there.
And I no longer think the cause is IPTG induction.
I don't use a lysis buffer---sonication.
OK, but what solution do you use when you resuspend the cell pellet ? I presume that's what you load the column with. If it's the same as the washbuffer, it looks OK, especially if the [NaCl] is about 150 mM.