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HIS-tagged protein purification


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#1 sashacat

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Posted 07 June 2011 - 06:00 AM

I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

#2 protolder

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Posted 15 June 2011 - 10:06 PM

I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

Hola as the days run and you havenīt any answer iīll give you my opinion. For me itīs strange that your protein doesnīt bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

#3 sashacat

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Posted 23 June 2011 - 05:29 AM


I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

Hola as the days run and you havenīt any answer iīll give you my opinion. For me itīs strange that your protein doesnīt bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

The binding buffer is 20 mM HEPES pH=8.0, 500mM NaCl, 10mM imidazol. The column does stay blue after the ran. Given that the protein is monomeric it really puzzles me. I suppose I can try phosphate buffer.

#4 sashacat

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Posted 23 June 2011 - 10:45 AM



I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

Hola as the days run and you havenīt any answer iīll give you my opinion. For me itīs strange that your protein doesnīt bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

The binding buffer is 20 mM HEPES pH=8.0, 500mM NaCl, 10mM imidazol. The column does stay blue after the ran. Given that the protein is monomeric it really puzzles me. I suppose I can try phosphate buffer.

Also I tried purifying a different protein at the exact same conditions and it worked well.

#5 Bluefunk

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Posted 25 June 2011 - 12:45 PM




I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

Hola as the days run and you havenīt any answer iīll give you my opinion. For me itīs strange that your protein doesnīt bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

The binding buffer is 20 mM HEPES pH=8.0, 500mM NaCl, 10mM imidazol. The column does stay blue after the ran. Given that the protein is monomeric it really puzzles me. I suppose I can try phosphate buffer.

Also I tried purifying a different protein at the exact same conditions and it worked well.


Try removing the imidazole from your binding buffer - that's all I got. If it still doesn't bind, I'm not convinved there's a his-tag there. Do a anti-His western blot on the crude extract to be sure?

#6 sashacat

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Posted 30 June 2011 - 07:24 AM





I am working on purification of his tagged protein. I am tracking the overexpressed protein by activity. So far the protein goes straight through the Ni2+ column. Does anyone know if adding of polygylcine linker between the protein and the histag would improve binding?

Hola as the days run and you havenīt any answer iīll give you my opinion. For me itīs strange that your protein doesnīt bound having a 6His tag. The colum remains blue after sample pass?. I would revise any of the incompatibilities of the column with sample buffer components before to construct a longer arm. because you could have the same problem,Buena suerte

The binding buffer is 20 mM HEPES pH=8.0, 500mM NaCl, 10mM imidazol. The column does stay blue after the ran. Given that the protein is monomeric it really puzzles me. I suppose I can try phosphate buffer.

Also I tried purifying a different protein at the exact same conditions and it worked well.


Try removing the imidazole from your binding buffer - that's all I got. If it still doesn't bind, I'm not convinved there's a his-tag there. Do a anti-His western blot on the crude extract to be sure?


I did in gel his tag staining using his tag stain from invitrogen and it shows that there is no his tag present. It is odd because I have the activity associated with the enzyme, but not his tag. Can it be that only his tag is being degraded or what else should I try to prevent histag degradation?

#7 almost a doctor

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Posted 30 June 2011 - 08:20 AM

So you know your protein of interest is there, but you can detect the his-tag? I'll recommend re-sequencing your expresion clone to confimr the His-tag is actually there, and in frame. I had this problem in the past, was unable to purify my protein through a His column despite being able to detect it on western blot (with protein specific antibodies). When I did an anti-his western I couldnt see anything. I then went back to my sequence,and found out that the His-tag was not in frame and therefore I did not have 6-His at the end of my protein.

Hope this helps.

#8 Bea Kerr

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Posted 07 July 2011 - 10:59 PM

I had a similar problem with one of my proteins years ago. I don't know which expression vector you are using but the His-tag is usually already in frame with the start codon of the vector so that shouldn't be an issue. With my protein, I concluded that the His-tag was folded somehow inside the protein and was thus not exposed - could not bind to Ni2+ IMAC column or react with anti-His antibody on Western blot. You could try purifying your protein under denaturing conditions which will expose the hidden his-tag for easier purification using IMAC - but then you obviously need to re-fold which will take some time to optimise. Can you use another method of purification such as size-exclusion or the like?

#9 sashacat

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Posted 12 July 2011 - 12:17 PM

I had a similar problem with one of my proteins years ago. I don't know which expression vector you are using but the His-tag is usually already in frame with the start codon of the vector so that shouldn't be an issue. With my protein, I concluded that the His-tag was folded somehow inside the protein and was thus not exposed - could not bind to Ni2+ IMAC column or react with anti-His antibody on Western blot. You could try purifying your protein under denaturing conditions which will expose the hidden his-tag for easier purification using IMAC - but then you obviously need to re-fold which will take some time to optimise. Can you use another method of purification such as size-exclusion or the like?



The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?

thanks for the post. Misery loves company.

#10 qzlabs

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Posted 12 July 2011 - 01:33 PM


I had a similar problem with one of my proteins years ago. I don't know which expression vector you are using but the His-tag is usually already in frame with the start codon of the vector so that shouldn't be an issue. With my protein, I concluded that the His-tag was folded somehow inside the protein and was thus not exposed - could not bind to Ni2+ IMAC column or react with anti-His antibody on Western blot. You could try purifying your protein under denaturing conditions which will expose the hidden his-tag for easier purification using IMAC - but then you obviously need to re-fold which will take some time to optimise. Can you use another method of purification such as size-exclusion or the like?



The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?

thanks for the post. Misery loves company.


You are right. On a SDS gel, proteins should be denatured and his tag should be exposed. Are you saying that the his tag is confirmed by DNA sequencing and is in frame with your insert? If that is the case, the only possiblity is that the N terminal of your fusion protein is truncated by the cell. I had one case like that. A c-terminal HIS tag will solve the problem. I suggest you should try our new bioreactors to get higher yield of your proteins so you do not need western to see your protein. Please check out our webpage at qizhonglabs.com for more details.
For higher yield and more protein, check out the new bioreactors at https://qizhonglabs.com/main/.

#11 tientranle

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Posted 02 August 2011 - 01:26 AM

Hi everyone, It's the first time I've joint the forum. I am now trying to purify my HIS-tagged protien. But I have a problem. After eluting, I did electrophoresis on SDS-PAGE. The result seems very good, there's just a band of interest. But after eluting step, I eliminated immidazole by using the PD-10 desalting column and then did electrophoresis again. It's so bad because many other bands appear. I did every step very carefully of course. There is anyone can tell me how to improve the bad result. Thanks sincerely for your kind help.

Edited by tientranle, 02 August 2011 - 03:21 AM.


#12 Bea Kerr

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Posted 08 September 2011 - 08:42 PM

#9 sashacat:

The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?

thanks for the post. Misery loves company.
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I wasn't getting any binding to the column so I suppose there was not protein when I ran my elution on SDS-PAGE/WB. Any luck until now with that protein?

#13 Adrian K

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Posted 09 September 2011 - 10:13 AM


The protein is very difficult to purify which is why I decided to go with his tag to begin with. The odd thing is that I attached the his-tag with all the different linkers in between including a long glycine polylinker and it won't bind. Also wouldn't protein be completely unfolded and thus histag exposed when you do SDS PAGE gel before you do Western blot?

thanks for the post. Misery loves company.


Before further troubleshooting, had you tried using your whole "cloned and expressed E. coli cells" (not the purified protein) and run SDS-PAGE & WB? DO you see your wanted band in this case?
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