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getting rid of imidazole in purified protein?

imidazole his-tag protein making antibody

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#1 fhn

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Posted 20 November 2011 - 03:55 PM

Hi guys,

I purified my his-tagged protein using qiagene Ni NTA . i eluted my protein in (50mM NaH2Po4, 300mM NaCl, 250mM imidazole). I will inject this protein to rabbit to make antibody. do I have to get rid of imidazole, if so, should I dialyse it? please help me.

Thank you in advance,
Farshad

#2 protolder

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Posted 20 November 2011 - 11:49 PM

Hola I think it´s be better inoculate your sample without imidazole, as far as I know you have to concentrate your positive fractions in order to inject an smal volume to the animals so, by the way if you concentrate by ultrafiltration dialyze in the ultrafiltration device your sample. For instance you have 15ml of sample and need concentrate to 2 ml for inject, so ultrafiltrate to the less volume possible and dilute again with your buffer at 3 volumes so making twice this you have at the end an imidazole conc or 25-30mM less toxic.or dialyze in a high volume in dyalisys bag and concentrate afterwards.lethal dose of imidazole in rat about 1g/k. Buena suerte
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#3 fhn

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Posted 22 November 2011 - 08:52 AM

Thanks Veteran, I did dialysis and i will inject my protein soon.




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