TAP Tagged Affinity Purification + Sticking to IgG-seph - (Sep/27/2004 )
Dear,
We are using as an initial step to capture our protein complexes(from plant cell cultures) Ig-G sepharose6 Fast Flow(Amersham Biosciences).
The tagged protein we're trying to capture has a TEV-cleaving site.
And we believe that even after TEV-cleavage still a lot of our protein sticks to the affinity-resin.
The evidence for this is after cleaving by boiling the resin in SDS-sample buffer something like 50% is found back.
Are you encountering the same problems?
And do you have some suggestions to solve this problem?
Yours sincerely,
Geert Persiau
VIB-Plant Systems Biology
--
We are encountering the same problem and are also using TAP tagging. No suggestions yet, but maybe soon. 
I am getting in the same problem, and the beads I used are also from Amersham.
Does anybody have any suggestions?
Sincerely yours,
fn17
We are using as an initial step to capture our protein complexes(from plant cell cultures) Ig-G sepharose6 Fast Flow(Amersham Biosciences).
The tagged protein we're trying to capture has a TEV-cleaving site.
And we believe that even after TEV-cleavage still a lot of our protein sticks to the affinity-resin.
The evidence for this is after cleaving by boiling the resin in SDS-sample buffer something like 50% is found back.
Are you encountering the same problems?
And do you have some suggestions to solve this problem?
Yours sincerely,
Geert Persiau
VIB-Plant Systems Biology
--
Hi,
We are having the same problem. We are trying to purify TAP tagged proteins using IgG Sepharose 6 Fast Flow. The tagged protein has a TEV cleavage site and after cleavage we would like to capture the cleaved protein product, however, the majority of the protein is stuck to the column after it is cut by the enzyme and we cannot get it off apart from boiling the sample in sample loading buffer containing SDS. Any suggestions to solving this problem would be greatly appreciated. Thanks in advance for your help.
RDP
Hi,
I Know from other people experience that TAP-tag is a quite unefficient system to purify protein complexes. TEV claveage and the other purification steps are triky, you have to set up the conditions for your protein and use huge amounts of extract to overcome the uneficiency. This huge amount of extract are esay to get from yeast but very dificult to get from mammalian cell culture. That was what I heard from friends from the EMBL where they developed this system. So I tried to use one or two immunopurifications just with FLAG tag or with HA tag. I saw that just one immunopurification step was enough. Maybe in parallel you can try with other tag different to TAP-tag and see.
Regards,
Antonio
Hi Antonio,
Thanks. We are in the process of trying Flag as well.
Best,
RDP
I Know from other people experience that TAP-tag is a quite unefficient system to purify protein complexes. TEV claveage and the other purification steps are triky, you have to set up the conditions for your protein and use huge amounts of extract to overcome the uneficiency. This huge amount of extract are esay to get from yeast but very dificult to get from mammalian cell culture. That was what I heard from friends from the EMBL where they developed this system. So I tried to use one or two immunopurifications just with FLAG tag or with HA tag. I saw that just one immunopurification step was enough. Maybe in parallel you can try with other tag different to TAP-tag and see.
Regards,
Antonio