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His-tag purification and his-antibody for western


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3 replies to this topic

#1 bcdmgou

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Posted 10 January 2002 - 01:48 AM

Dear all
I met a problem in purification my protein expressed from pichia KM71 cell. it is a novel membrane protein and I can detect the expression in the crude membrane of km71. After purification with the Qiagen Ni-NTA kit, I got a very sharp band, a little smaller than the expected size. To my surprise, the band can not be detected with anti-His in Western. WHY??
Hoping get some information from any one.
Yours
bcdmgou

#2 ragno

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Posted 13 February 2002 - 09:52 AM

Hi,
I think, may be, that HIS sequence could be eliminate during expression.
You can see protein after purification with coomassie but not detected with anti-HIS antibody.
good luke
michel

#3 volkandemir

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Posted 12 November 2003 - 07:37 PM

do you make western blot at native conditions or denaturent conditions, hence if you make it in native conditions and if histag was stucked in the protein, it may not be detected. And are you sure that your second anribody recognizes primary antibody, you can test this step or may be you shoul optimizze western blot conditions....

Edited by volkandemir, 12 November 2003 - 07:37 PM.


#4 Oxygen

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Posted 03 May 2014 - 05:44 AM

Dear all
I met a problem in purification my protein expressed from pichia KM71 cell. it is a novel membrane protein and I can detect the expression in the crude membrane of km71. After purification with the Qiagen Ni-NTA kit, I got a very sharp band, a little smaller than the expected size. To my surprise, the band can not be detected with anti-His in Western. WHY??
Hoping get some information from any one.
Yours
bcdmgou

Hi,

 

I had met with similar problem with his-tag protein, but I've no solution for it. I wonder if you had any with your experience? My issue is, I could detect protein using western when I use induced cell lysate, and signal disappears when I use NiNTA purified protein. Any suggestion? also the band size get vary when protein was in cell lysate and after purification? Weird this is'nt it ?


Edited by Oxygen, 03 May 2014 - 05:45 AM.





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