Imidazole - levels and toxicity (Dec/28/2005 )
Someone else posted this question before and never had it answered. Now I find myself wondering the same thing. When doing His tag purification, many elution buffers use imidazole. Does anyone know how much is ok to inject (when injecting the proteins) in rabbits for antibody generation before it gets too toxic for the poor bunny?
this might be a dumb question, but isn't the imidazole removed during dialysis?
perhaps your workflow is different for your protocol, but this is how we do it:
2. dialysis (slide-a-lyzer)
3. concentration (amicon microconcentrators)
steps 2 and 3 should both help to remove imidazole? this is what I have always thought, but I guess I don't really know....unless you go directly from step 1 to induction in the rabbit?
Hey, it was tickling my brain and I had a few minutes so I went and found it...this is what I found on Qiagen's protein purification FAQ's:
Imidazole does not interfere with most downstream applications and therefore does not need to be removed. If it is necessary to remove the imidazole (e.g., for some sensitive enzyme assays), it can be easily achieved by one of three methods: • Dialysis • Precipitation (e.g., ammonium sulfate) • Ultrafiltration
also, an MSDS gave me this: CAS# 288-32-4:
Oral, mouse: LD50 = 880 mg/kg;
Oral, rat: LD50 = 220 mg/kg;
So, perhaps it is unknown for rabbits at this point? there was no SubCu information available
Thanks aimikins, I found this also here:
* Oral mouse LD50 1880 mg/kg
* Intraperitoneal mouse LD50 300 mg/kg
* Intravenous mouse LD50 475 mg/kg
* Subcutaneous rat LD50 626 mg/kg
(I would guess that the 1880 is a mistype on their part)
I haven't done dialysis yet. I am new to protein purification so I wasn't sure if it was possible to go directly to rabbit or not.
oh, hey, if you like Pierce's stuff, check out their slide-a-lyzer; it is fast and easy and much less cumbersome than any method I've tried...we have used it on many protein preps; our downstream stuff involves tissue culture cells (and we used to do animal work)
Thanks. The pierce dialysers look pretty nifty. Do the buffers come with it or is that something you need to have on hand? In fact, since you have done animal experiments before, allow me to pick your brain for a sec...what buffer do you use for animal work? Thanks for all your help so far btw, you have helped me a lot.