Re: [ccp4bb] Difficult purification with imac columns
I had a similar case before. I could not find notes for the details. 1. During the lysis, add protein amine sulfate and spin down with the speed up to 10K rpm (normally 8k) for 30 mins. 2. adding polysaccharide (1~ 5 %) as additive to adjust nonspecific bind. For polysaccharides, I tried several kinds of them ordered from Sigma. I forgot which one was the best (sorry). 3. use ionic columns ( anionic + cationic columns, chained them together) at 4C. 4. Then, used Co-Column (not, NTA,) for further purification. ... The final purity could reach 95%. However, l lost a lot of protein during purification. On Fri, Sep 15, 2017 at 4:53 AM, Narayanan Ramasubbu < ramas...@sdm.rutgers.edu> wrote: > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is > the full length protein. The difficulty us that it strongly binds to the > resin with or without his.tag. Changing the resin to acrylamide did not > help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone -- Kevin Jin Sharing knowledge each other is always very joyful.. Website: http://www.jinkai.org/
Re: [ccp4bb] Difficult purification with imac columns
Hi Narayanan, This doesn't address your question; may be you could go-around this problem by using a different purification tag., say like GST?!! Good Luck, Partha On Fri, Sep 15, 2017 at 7:54 AM Narayanan Ramasubbu < ramas...@sdm.rutgers.edu> wrote: > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is > the full length protein. The difficulty us that it strongly binds to the > resin with or without his.tag. Changing the resin to acrylamide did not > help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone
Re: [ccp4bb] Difficult purification with imac columns
Hi Narayanan,We had similar problems with a membrane protein. It did bind Ni-NTA resin strongly. We washed out the resin (Ni-NTA) with high imidazole to sealer all other proteins and eluted out protein with very small concentration of SDS at room temp with shaking. Please read our paper in- Huang et al., Biochemistry, 49, 1115-1126, 2010Hope this procedure can help you too.Smita On Friday, September 15, 2017 6:54 AM, Narayanan Ramasubbu wrote: Hi. We are working on a periplasmic protein that breaks naked glycans in peptidoglycans. There is truncated structure available but our target is the full length protein. The difficulty us that it strongly binds to the resin with or without his.tag. Changing the resin to acrylamide did not help. Has anyone come across similar problem and how was it resolved. The pdb structure is the catalytic domain and mussing a region that, in my opinion, binds to the resin. Thank you in advance Sent from my iPhone
Re: [ccp4bb] Difficult purification with imac columns
Hi This is a multidimensional problem, just like any other tricky protein purification. 1) IMAC columns are also ion exchangers (of both polarities!) so make sure you have adequate salt. 2) IMAC resins can be polysaccharide-based (agarose) and affinity to sugars can be an issue. Consider adding a 'mock substrate' (basically a high level of some inexpensive sugar). 3) Even if you cannot use IMAC there are other options, people purify native proteins in a 'generic' manner all the time :) 4) definitely a case for trying other resins. Especially consider resins with pentavalent chelation - they have rather different properties from resins based on tri (IDA) or tetra (NTA) chelators. 5) as others have mentioned, you may have a problem of the protein as such - can you assess aggregation state before purification (e.g. SEC of clarified lysate followed by activity assay). Artem - Cosmic Cats approve of this message On Fri, Sep 15, 2017 at 7:53 AM, Narayanan Ramasubbu < ramas...@sdm.rutgers.edu> wrote: > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is > the full length protein. The difficulty us that it strongly binds to the > resin with or without his.tag. Changing the resin to acrylamide did not > help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone
Re: [ccp4bb] Difficult purification with imac columns
Dear Narayanan Which kind of resin are you using ? In my lab we work with proteins that bind dextran which is often used as crosslinker in chromatography resins. Crosslinked resins have a highly reticulated structure made of sugars that can be quite similar to peptidoglycan... As a test, you could try to inject your protein in the presence of glucose (or another sugar) to compete with this interaction. Changing pH and NaCl concentration might help too. Good luck, Gianluca. Il 15/Set/2017 01:54 PM, "Narayanan Ramasubbu" ha scritto: > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is > the full length protein. The difficulty us that it strongly binds to the > resin with or without his.tag. Changing the resin to acrylamide did not > help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone
Re: [ccp4bb] Difficult purification with imac columns
change buffer，or use different resin matrix from another compny 发自网易邮箱大师 在2017年09月15日 19:53，Narayanan Ramasubbu 写道： Hi. We are working on a periplasmic protein that breaks naked glycans in peptidoglycans. There is truncated structure available but our target is the full length protein. The difficulty us that it strongly binds to the resin with or without his.tag. Changing the resin to acrylamide did not help. Has anyone come across similar problem and how was it resolved. The pdb structure is the catalytic domain and mussing a region that, in my opinion, binds to the resin. Thank you in advance Sent from my iPhone
Re: [ccp4bb] Difficult purification with imac columns
Hi! Maybe you protein is present as soluble microaggregates and gets stuck on the resin, without really 'binding' to it. I would spin the lysate in an ultracentrifuge 1h @ 50 k or so, to see if there is anything left in the supernant afterwards. And then make decisions based on that outcome. best, sebastian > On 15. Sep 2017, at 13:53, Narayanan Ramasubbu > wrote: > > Hi. We are working on a periplasmic protein that breaks naked glycans in > peptidoglycans. There is truncated structure available but our target is the > full length protein. The difficulty us that it strongly binds to the resin > with or without his.tag. Changing the resin to acrylamide did not help. > Has anyone come across similar problem and how was it resolved. > The pdb structure is the catalytic domain and mussing a region that, in my > opinion, binds to the resin. > Thank you in advance > Sent from my iPhone
[ccp4bb] Difficult purification with imac columns
Hi. We are working on a periplasmic protein that breaks naked glycans in peptidoglycans. There is truncated structure available but our target is the full length protein. The difficulty us that it strongly binds to the resin with or without his.tag. Changing the resin to acrylamide did not help. Has anyone come across similar problem and how was it resolved. The pdb structure is the catalytic domain and mussing a region that, in my opinion, binds to the resin. Thank you in advance Sent from my iPhone
