Re: [ccp4bb] gst-tag protein purify problem
Forgot to add, a 150aa dna binding protein should only need a minimal hexahistidine tagthese proteins are typically very soluble and express well. Adding a GST or sumo tag is overkill. Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. --- On Sat, 8/6/11, Carlos Kikuti kik...@gmail.com wrote: From: Carlos Kikuti kik...@gmail.com Subject: Re: [ccp4bb] gst-tag protein purify problem To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, August 6, 2011, 5:13 PM What happens if you load your elution fraction into a size exclusion column? If your protein of interest comes in the void volume together with most of its contaminants, you'd better test a different construct, and that's much more than only changing the tag from N- to C-terminus. Sumo and GST might be solubilizing things that shouldn't really be soluble... Carlos Em 06/08/2011, às 16:43, Paul Kraft escreveu: Hi Lisa, Have you compared your yield of purified protein of soluble protein per gram of pellet, 4M Guanidine solubilized pellet (wash and elute from Ni column with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total protein in the pellet on a page gel? The main reason I would think you get high background of cellular proteins on you Ni purification is because your yield is too low (I know obvious..). There are a variety of methods to increase yield like expressing at RT (assuming your expressing in E.coli), or switching to yeast, insect, or mamallian cells if your protein is of human origin. The most helpful and easiest method to try first (after trying low temp), would be switching the tag from N to C terminus or vice versa. Otherwise switch to a thermophile version of your protein if possible (and try cysteine mutants or other bacterial organisms for source the source gene). Paul ps one thing I forgot, you mention it is a DNA binding protein, solublizing in 2M NaCl is much better than 1M NaCl, you could just be losing it...the protein that is :-) Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. From: LISA science...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, August 4, 2011 11:51 AM Subject: [ccp4bb] gst-tag protein purify problem hi guys, I have a DNA binding protein and expressed the DNA binding domain (150 aa) with his-sumo tag or gst tag at the n-terminal. I tried to purified it with Ni column or Gst column separately. But purity is lower than 50% after Ni or GST column. This protein only stable with 1M Nacl or higher. I worked on it almost half year. But I still can not get the pure protein. please give me some suggestions. Thank you. Lisa
Re: [ccp4bb] gst-tag protein purify problem
Hi Lisa, Have you compared your yield of purified protein of soluble protein per gram of pellet, 4M Guanidine solubilized pellet (wash and elute from Ni column with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total protein in the pellet on a page gel? The main reason I would think you get high background of cellular proteins on you Ni purification is because your yield is too low (I know obvious..). There are a variety of methods to increase yield like expressing at RT (assuming your expressing in E.coli), or switching to yeast, insect, or mamallian cells if your protein is of human origin. The most helpful and easiest method to try first (after trying low temp), would be switching the tag from N to C terminus or vice versa. Otherwise switch to a thermophile version of your protein if possible (and try cysteine mutants or other bacterial organisms for source the source gene). Paul ps one thing I forgot, you mention it is a DNA binding protein, solublizing in 2M NaCl is much better than 1M NaCl, you could just be losing it...the protein that is :-) Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. From: LISA science...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, August 4, 2011 11:51 AM Subject: [ccp4bb] gst-tag protein purify problem hi guys, I have a DNA binding protein and expressed the DNA binding domain (150 aa) with his-sumo tag or gst tag at the n-terminal. I tried to purified it with Ni column or Gst column separately. But purity is lower than 50% after Ni or GST column. This protein only stable with 1M Nacl or higher. I worked on it almost half year. But I still can not get the pure protein. please give me some suggestions. Thank you. Lisa
Re: [ccp4bb] gst-tag protein purify problem
What happens if you load your elution fraction into a size exclusion column? If your protein of interest comes in the void volume together with most of its contaminants, you'd better test a different construct, and that's much more than only changing the tag from N- to C-terminus. Sumo and GST might be solubilizing things that shouldn't really be soluble... Carlos Em 06/08/2011, às 16:43, Paul Kraft escreveu: Hi Lisa, Have you compared your yield of purified protein of soluble protein per gram of pellet, 4M Guanidine solubilized pellet (wash and elute from Ni column with 8M urea .5M imidazole..otherwise the gel with run crappy), and and total protein in the pellet on a page gel? The main reason I would think you get high background of cellular proteins on you Ni purification is because your yield is too low (I know obvious..). There are a variety of methods to increase yield like expressing at RT (assuming your expressing in E.coli), or switching to yeast, insect, or mamallian cells if your protein is of human origin. The most helpful and easiest method to try first (after trying low temp), would be switching the tag from N to C terminus or vice versa. Otherwise switch to a thermophile version of your protein if possible (and try cysteine mutants or other bacterial organisms for source the source gene). Paul ps one thing I forgot, you mention it is a DNA binding protein, solublizing in 2M NaCl is much better than 1M NaCl, you could just be losing it...the protein that is :-) Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. From: LISA science...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, August 4, 2011 11:51 AM Subject: [ccp4bb] gst-tag protein purify problem hi guys, I have a DNA binding protein and expressed the DNA binding domain (150 aa) with his-sumo tag or gst tag at the n-terminal. I tried to purified it with Ni column or Gst column separately. But purity is lower than 50% after Ni or GST column. This protein only stable with 1M Nacl or higher. I worked on it almost half year. But I still can not get the pure protein. please give me some suggestions. Thank you. Lisa
[ccp4bb] gst-tag protein purify problem
hi guys, I have a DNA binding protein and expressed the DNA binding domain (150 aa) with his-sumo tag or gst tag at the n-terminal. I tried to purified it with Ni column or Gst column separately. But purity is lower than 50% after Ni or GST column. This protein only stable with 1M Nacl or higher. I worked on it almost half year. But I still can not get the pure protein. please give me some suggestions. Thank you. Lisa
