Re: [ccp4bb] very high concentration of protein
Dear Peter, it's a common phenomenon to create protein concentration gradients inside a protein concentrator. Simply take it all out of the concentrator, put it in a separate tube, and mix thoroughly/vortex. The 'slime' may very well redissolve and you'll a homogeneous distribution. What you describe happens a lot to us with proteins that don't precipitate and that crystallize afterwards. Cheers Filip Van Petegem On Thu, Jul 2, 2009 at 8:53 AM, peter hudson wrote: > Hello all > > I am working with a small protein-protein complex. This complex express > quite well . I purify in a buffer of pH=9.0 with 150mM NaCl and 1% of > glycerol and able to concentrate upto 20 mg per ml. I have a two clones of > this protein complex. One is N-terminal His tagged and another C-terminal > His-tagged. While concentration of the N-terminal His tagged protein in > cnetricon it forms yellow color slimy deposition on the surface of membrane. > while C-terminal His tagged protein does form very highly viscous layere at > the surface of membrane but it is completly colourless.I aliquate the > concentrated protein by pippetting into different aliquate rather than > collection of whole protein by centrifugation. if i check the concentration > of the last aliqoute(which isbottommost viscous protein part) both prtoein > complex, it shows very high concentration compared to the first fraction. I > have not done DLS. > Is my both C-terminal His tagged tagged as well as N-terminal His tagged > protein are forming soluble aggregates. > > I would appreciate the help. > > Thanks in advance > > Peter > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] very high concentration of protein
Peter, If I understand what you are saying - then it is very likely that your protein forms aggregates. Whether this happens on concentration or not is unknown because concentration may simply bring the pre-existing aggregates to the membrane. You can try to make concentration process 'easy' on the protein by avoiding local over-concentration (formation of a gradient in the centrifugal device). This can be done by using the inverted (upward-concentraring) concentrators (such as the Millipore variety - they don't eliminate the gradient completely but are nevertheless a step above the downward-concentrating versions) or even better by using a stirred cell etc. It is generally a good idea to test your protein's oligomeric state and homogeneity before and after concentration using e.g. analytical size exclusion or light scattering (or analytical centrifugation, or whatever else you may have in the lab). Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of peter hudson Sent: Thursday, July 02, 2009 10:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] very high concentration of protein Hello all I am working with a small protein-protein complex. This complex express quite well . I purify in a buffer of pH=9.0 with 150mM NaCl and 1% of glycerol and able to concentrate upto 20 mg per ml. I have a two clones of this protein complex. One is N-terminal His tagged and another C-terminal His-tagged. While concentration of the N-terminal His tagged protein in cnetricon it forms yellow color slimy deposition on the surface of membrane. while C-terminal His tagged protein does form very highly viscous layere at the surface of membrane but it is completly colourless.I aliquate the concentrated protein by pippetting into different aliquate rather than collection of whole protein by centrifugation. if i check the concentration of the last aliqoute(which isbottommost viscous protein part) both prtoein complex, it shows very high concentration compared to the first fraction. I have not done DLS. Is my both C-terminal His tagged tagged as well as N-terminal His tagged protein are forming soluble aggregates. I would appreciate the help. Thanks in advance Peter
[ccp4bb] very high concentration of protein
Hello all I am working with a small protein-protein complex. This complex express quite well . I purify in a buffer of pH=9.0 with 150mM NaCl and 1% of glycerol and able to concentrate upto 20 mg per ml. I have a two clones of this protein complex. One is N-terminal His tagged and another C-terminal His-tagged. While concentration of the N-terminal His tagged protein in cnetricon it forms yellow color slimy deposition on the surface of membrane. while C-terminal His tagged protein does form very highly viscous layere at the surface of membrane but it is completly colourless.I aliquate the concentrated protein by pippetting into different aliquate rather than collection of whole protein by centrifugation. if i check the concentration of the last aliqoute(which isbottommost viscous protein part) both prtoein complex, it shows very high concentration compared to the first fraction. I have not done DLS. Is my both C-terminal His tagged tagged as well as N-terminal His tagged protein are forming soluble aggregates. I would appreciate the help. Thanks in advance Peter
