Hi, thanks for your answers. Concerning the HABA removal... Well, it turned out to be rather my fault. Since I was using four different solutions I used all the lines, but I didn't purge it before run because I wanted to save the elution buffer and I didn't realize there's the HABA in between the pump and the mixer. I'll try it again, probably next week and I'll see whether that was the problem.
myc-tag was used previously on the C-terminus as well and it was succesful with purification. The problem is, that I tried other protein, which worked previously with StrepTactin in batch method and in my hands it didn't work (I'm repeating the PAGE and bloting, because it didn't work very well). So I'll have to try it the batch methods first time.
Hi, thanks for your answers. Concerning the HABA removal... Well, it turned out to be rather my fault. Since I was using four different solutions I used all the lines, but I didn't purge it before run because I wanted to save the elution buffer and I didn't realize there's the HABA in between the pump and the mixer. I'll try it again, probably next week and I'll see whether that was the problem.
myc-tag was used previously on the C-terminus as well and it was succesful with purification. The problem is, that I tried other protein, which worked previously with StrepTactin in batch method and in my hands it didn't work (I'm repeating the PAGE and bloting, because it didn't work very well). So I'll have to try it the batch methods first time.
In my experience, the problem of binding of the tag to the Strep-Tactin column was solved after introducing a linker between the Strep-II tag and the C-terminus of the protein in question. Thorough washing between different steps and during the regeneration has always primary importance.
In my experience, the problem of binding of the tag to the Strep-Tactin column was solved after introducing a linker between the Strep-II tag and the C-terminus of the protein in question. Thorough washing between different steps and during the regeneration has always primary importance.
Yeah, unfortunately I tried batch purification with the MacroPrep yesterday and it didn't work :-/ Now I'm doing blot of the positive controls, so I'll see if it's me or just the protein...
Yeah, unfortunately I tried batch purification with the MacroPrep yesterday and it didn't work :-/ Now I'm doing blot of the positive controls, so I'll see if it's me or just the protein...
Good luck. By the way, what kind of protein are you trying to purify? Cytoplasmic? Does it have properties that would make it aggregate/precipitate out? I assume you clarify your lysates by centrifugation prior to loading on the column. Is it possible your protein is in the pellet fraction? Do you run out input, flowthrough, washes and elution fractions on SDS-PAGE and track your protein by western blot through the purification process?
Good luck. By the way, what kind of protein are you trying to purify? Cytoplasmic? Does it have properties that would make it aggregate/precipitate out? I assume you clarify your lysates by centrifugation prior to loading on the column. Is it possible your protein is in the pellet fraction? Do you run out input, flowthrough, washes and elution fractions on SDS-PAGE and track your protein by western blot through the purification process?
If all it is is HABA in your lines, hopefully a more thorough wash will solve the issue. If you are still unable to see binding, you may need to think about switching the tag to the other terminus or adding a linker. Maybe you'll have better luck with batch purification.
If all it is is HABA in your lines, hopefully a more thorough wash will solve the issue. If you are still unable to see binding, you may need to think about switching the tag to the other terminus or adding a linker. Maybe you'll have better luck with batch purification.
The protein was re-cloned in the meanwhile with GFP in between my protein and the Strep-tag and this construct worked quite nicely. We stil had some contaminating proteins though.
The protein was re-cloned in the meanwhile with GFP in between my protein and the Strep-tag and this construct worked quite nicely. We stil had some contaminating proteins though.
Well, the localization seems to be little tricky, it's probably associated with something like cell wall maybe... (unextractable with 1% Triton), but the part I'm working with is soluble protein. I put everything on the western (or when I measure activity) and it's clearly in the unbound while nothing in elution.
Well, the localization seems to be little tricky, it's probably associated with something like cell wall maybe... (unextractable with 1% Triton), but the part I'm working with is soluble protein. I put everything on the western (or when I measure activity) and it's clearly in the unbound while nothing in elution.
If you have the problem still now with protein binding to column then...check the protein sample pH before loading to the column. I don't know what type of buffer and pH of buffer you are using for the protein extraction. I faced similar problem with my protein. Then I adjusted the pH (final pH 8 ) of the crude protein sample adding some drops of dilute NaOH. I always adjust the protein sample pH 8 as I am using binding buffer and Elution buffer with pH 8. In my protein the tag is at the N-terminal end and its seems ok. The regeneration of column is nice with 0.5 M NaOH....
If you have the problem still now with protein binding to column then...check the protein sample pH before loading to the column. I don't know what type of buffer and pH of buffer you are using for the protein extraction. I faced similar problem with my protein. Then I adjusted the pH (final pH 8 ) of the crude protein sample adding some drops of dilute NaOH. I always adjust the protein sample pH 8 as I am using binding buffer and Elution buffer with pH 8. In my protein the tag is at the N-terminal end and its seems ok. The regeneration of column is nice with 0.5 M NaOH....
Hi,
thanks for your answers.
Concerning the HABA removal... Well, it turned out to be rather my fault. Since I was using four different solutions I used all the lines, but I didn't purge it before run because I wanted to save the elution buffer and I didn't realize there's the HABA in between the pump and the mixer. I'll try it again, probably next week and I'll see whether that was the problem.
myc-tag was used previously on the C-terminus as well and it was succesful with purification. The problem is, that I tried other protein, which worked previously with StrepTactin in batch method and in my hands it didn't work (I'm repeating the PAGE and bloting, because it didn't work very well). So I'll have to try it the batch methods first time.
Hi,
thanks for your answers.
Concerning the HABA removal... Well, it turned out to be rather my fault. Since I was using four different solutions I used all the lines, but I didn't purge it before run because I wanted to save the elution buffer and I didn't realize there's the HABA in between the pump and the mixer. I'll try it again, probably next week and I'll see whether that was the problem.
myc-tag was used previously on the C-terminus as well and it was succesful with purification. The problem is, that I tried other protein, which worked previously with StrepTactin in batch method and in my hands it didn't work (I'm repeating the PAGE and bloting, because it didn't work very well). So I'll have to try it the batch methods first time.
More
VOTE
In my experience, the problem of binding of the tag to the Strep-Tactin column was solved after introducing a linker between the Strep-II tag and the C-terminus of the protein in question. Thorough washing between different steps and during the regeneration has always primary importance.
In my experience, the problem of binding of the tag to the Strep-Tactin column was solved after introducing a linker between the Strep-II tag and the C-terminus of the protein in question. Thorough washing between different steps and during the regeneration has always primary importance.
More
VOTE
Yeah, unfortunately I tried batch purification with the MacroPrep yesterday and it didn't work :-/ Now I'm doing blot of the positive controls, so I'll see if it's me or just the protein...
Yeah, unfortunately I tried batch purification with the MacroPrep yesterday and it didn't work :-/ Now I'm doing blot of the positive controls, so I'll see if it's me or just the protein...
More
VOTE
Good luck. By the way, what kind of protein are you trying to purify? Cytoplasmic? Does it have properties that would make it aggregate/precipitate out? I assume you clarify your lysates by centrifugation prior to loading on the column. Is it possible your protein is in the pellet fraction? Do you run out input, flowthrough, washes and elution fractions on SDS-PAGE and track your protein by western blot through the purification process?
Good luck. By the way, what kind of protein are you trying to purify? Cytoplasmic? Does it have properties that would make it aggregate/precipitate out? I assume you clarify your lysates by centrifugation prior to loading on the column. Is it possible your protein is in the pellet fraction? Do you run out input, flowthrough, washes and elution fractions on SDS-PAGE and track your protein by western blot through the purification process?
More
VOTE
I used gel filtration chromatograpy and got pure protein.
I used gel filtration chromatograpy and got pure protein.
More
VOTE
If all it is is HABA in your lines, hopefully a more thorough wash will solve the issue. If you are still unable to see binding, you may need to think about switching the tag to the other terminus or adding a linker. Maybe you'll have better luck with batch purification.
If all it is is HABA in your lines, hopefully a more thorough wash will solve the issue. If you are still unable to see binding, you may need to think about switching the tag to the other terminus or adding a linker. Maybe you'll have better luck with batch purification.
More
VOTE
The protein was re-cloned in the meanwhile with GFP in between my protein and the Strep-tag and this construct worked quite nicely. We stil had some contaminating proteins though.
The protein was re-cloned in the meanwhile with GFP in between my protein and the Strep-tag and this construct worked quite nicely. We stil had some contaminating proteins though.
More
VOTE
Well, the localization seems to be little tricky, it's probably associated with something like cell wall maybe... (unextractable with 1% Triton), but the part I'm working with is soluble protein.
I put everything on the western (or when I measure activity) and it's clearly in the unbound while nothing in elution.
Well, the localization seems to be little tricky, it's probably associated with something like cell wall maybe... (unextractable with 1% Triton), but the part I'm working with is soluble protein.
I put everything on the western (or when I measure activity) and it's clearly in the unbound while nothing in elution.
More
VOTE
Hi Tomas,
If you have the problem still now with protein binding to column then...check the protein sample pH before loading to the column. I don't know what type of buffer and pH of buffer you are using for the protein extraction. I faced similar problem with my protein. Then I adjusted the pH (final pH 8 ) of the crude protein sample adding some drops of dilute NaOH. I always adjust the protein sample pH 8 as I am using binding buffer and Elution buffer with pH 8. In my protein the tag is at the N-terminal end and its seems ok. The regeneration of column is nice with 0.5 M NaOH....
Hi Tomas,
If you have the problem still now with protein binding to column then...check the protein sample pH before loading to the column. I don't know what type of buffer and pH of buffer you are using for the protein extraction. I faced similar problem with my protein. Then I adjusted the pH (final pH 8 ) of the crude protein sample adding some drops of dilute NaOH. I always adjust the protein sample pH 8 as I am using binding buffer and Elution buffer with pH 8. In my protein the tag is at the N-terminal end and its seems ok. The regeneration of column is nice with 0.5 M NaOH....
More
VOTE
To get rid of HABA, I always wash the column with 0.5M NaOH and then quickly equilibrate it with Tris-NaCl buffer, it works completely fine.
To get rid of HABA, I always wash the column with 0.5M NaOH and then quickly equilibrate it with Tris-NaCl buffer, it works completely fine.
More
VOTE
To get rid of HABA, I always wash the column with 0.5M NaOH and then quickly equilibrate it with Tris-NaCl buffer, it works completely fine.
To get rid of HABA, I always wash the column with 0.5M NaOH and then quickly equilibrate it with Tris-NaCl buffer, it works completely fine.
More
VOTE