HIS tag elution woes.. - (May/01/2007 )
I am currently using a coupled in vitro transcription translation system to make proteins for my work... messed about for over a year and managed to get soluble but non functional protein from bacterial expression..
i am working on some novel histone acetyltransferases, the first couple of in vitro TnT kits worked a treat, then the batch number changed and i started getting some exogenous acetylation..
The template i use is a PCR construct that has a 5' SP6 promoter-kozak sequence-hybridisation sequence..... all was working well with this method so i decided to add a HQ (HQHQHQ) affinity tag to the 3' and purify my protein thereby getting rid of any endogenous AT activity...the new 3' primer work well and protein is still made..
To purify i have used a paramagnetic nickel bead system from promega, i used a fluorescent- lysene - tRNA conjugate in the TnT reaction so the protein could be traced through the purification process (s35 substitute).
This is where my tail of woe begins, i have used 500 mm NaCl, 25 mm imidazole in all the wash steps (4 in total) which gets rid of most of the non specifics, then it comes to elution, i just cannot get one of my proteins to elute from the beads
I have titrated imidazole up to 1 molar, incubated for up to 30 minutes, used salt up to 1 molar and dropped the pH as low as 4 ... not a thing comes off... but put a little sds in there and there is the protein..
i dont really want to go down the denaturing route as refolding is a nightmare.. (yup tried that before)
I thought it would be fine to keep the protein on the beads and do the assays on bead... but core histones also stick to the bead...
Can anybody shed any light on this for me... we have a tight deadline for publication of this data, once we have these papaers i ca relax a little and spend some time cloning into the pING 19.1 vector and use a strep II tag
many thanks
i am working on some novel histone acetyltransferases, the first couple of in vitro TnT kits worked a treat, then the batch number changed and i started getting some exogenous acetylation..
The template i use is a PCR construct that has a 5' SP6 promoter-kozak sequence-hybridisation sequence..... all was working well with this method so i decided to add a HQ (HQHQHQ) affinity tag to the 3' and purify my protein thereby getting rid of any endogenous AT activity...the new 3' primer work well and protein is still made..
To purify i have used a paramagnetic nickel bead system from promega, i used a fluorescent- lysene - tRNA conjugate in the TnT reaction so the protein could be traced through the purification process (s35 substitute).
This is where my tail of woe begins, i have used 500 mm NaCl, 25 mm imidazole in all the wash steps (4 in total) which gets rid of most of the non specifics, then it comes to elution, i just cannot get one of my proteins to elute from the beads
I have titrated imidazole up to 1 molar, incubated for up to 30 minutes, used salt up to 1 molar and dropped the pH as low as 4 ... not a thing comes off... but put a little sds in there and there is the protein..
i dont really want to go down the denaturing route as refolding is a nightmare.. (yup tried that before)
I thought it would be fine to keep the protein on the beads and do the assays on bead... but core histones also stick to the bead...
Can anybody shed any light on this for me... we have a tight deadline for publication of this data, once we have these papaers i ca relax a little and spend some time cloning into the pING 19.1 vector and use a strep II tag
many thanks
May be non-specific sorbtion on beads? Try another matrix for purification like Ni-NTA resin , or another method for purification. For ex IEC ( if you know pI of your protein)
i am working on some novel histone acetyltransferases, the first couple of in vitro TnT kits worked a treat, then the batch number changed and i started getting some exogenous acetylation..
The template i use is a PCR construct that has a 5' SP6 promoter-kozak sequence-hybridisation sequence..... all was working well with this method so i decided to add a HQ (HQHQHQ) affinity tag to the 3' and purify my protein thereby getting rid of any endogenous AT activity...the new 3' primer work well and protein is still made..
To purify i have used a paramagnetic nickel bead system from promega, i used a fluorescent- lysene - tRNA conjugate in the TnT reaction so the protein could be traced through the purification process (s35 substitute).
This is where my tail of woe begins, i have used 500 mm NaCl, 25 mm imidazole in all the wash steps (4 in total) which gets rid of most of the non specifics, then it comes to elution, i just cannot get one of my proteins to elute from the beads
I have titrated imidazole up to 1 molar, incubated for up to 30 minutes, used salt up to 1 molar and dropped the pH as low as 4 ... not a thing comes off... but put a little sds in there and there is the protein..
i dont really want to go down the denaturing route as refolding is a nightmare.. (yup tried that before)
I thought it would be fine to keep the protein on the beads and do the assays on bead... but core histones also stick to the bead...
Can anybody shed any light on this for me... we have a tight deadline for publication of this data, once we have these papaers i ca relax a little and spend some time cloning into the pING 19.1 vector and use a strep II tag
many thanks
May be non-specific sorbtion on beads? Try another matrix for purification like Ni-NTA resin , or another method for purification. For ex IEC ( if you know pI of your protein)
i have also tried ni nta with the same results..
Well! What is about elution with Ni by EDTA. But NiEDTA complex will be on your tag. Is it bad for your further exp?