Re: [ccp4bb] Interesting DNA contamination
Hi, I found that my protein bound ethidium bromide in an agarose gel. I tested that by treating my protein with protease and DNAse in two different tubes and running a gel. The band in the agarose gel disappeared only when the protein was treated with protease. It is worth trying. I hope that helps, Chiara Pramod, You already got good suggestions on how to handle DNA contamination in protein preparations. Let me point out briefly that you haven't demonstrated yet that your contamination is DNA. I had the same observation when purifying UvsX. A very persistent and strong contamination in all my preps at ~500kb. To test weather it was DNA or RNA I boiled the protein 30 minutes and incubated it with DNAase and RNAse and result was the same. I concluded it was neither RNA nor DNA and continued as if nothing had happened. This publication is reporting the same observation: Formosa and Alberts (1986) Purification and characterization of the T4 bacteriophage uvsX protein. J Biol Chem. 1986 May 5;261(13):6107-18. If you ever find out what it is that runs like 500kb DNA on Agarose, please let me know. S. -- Date:Sat, 27 Jun 2015 04:39:45 +0100 From:Stefan Gajewski sgajew...@gmail.com mailto:sgajew...@gmail.com Subject: Re: Interesting DNA contamination Correction, I meant to say 0.5kb, not 500kb sorry for that. S. -- Date:Sat, 27 Jun 2015 16:52:06 + From:Rose, Peter pwr...@ucsd.edu mailto:pwr...@ucsd.edu Subject: Postdoctoral Fellows in Big Data/Structural Bioinformatics at University of California, San Diego Summary: We are looking for two highly motivated post-docs as part of our new project “Compressive Structural Bioinformatics” funded by the US National Institutes of Health (NIH) Big Data to Knowledge (BD2K) initiative. The Challenge: To enable efficient research on the rapidly growing number of 3D molecular structures of ever increasing size and complexity. Develop highly scalable 3D structural search, analysis, workflow, data-exchange, and visualization tools. Qualifications: Ph.D. in structural bioinformatics, structural biology, bioinformatics, computational biology or chemistry, computer science, or related discipline. Experience with scientific software development as demonstrated by publications or participation in open source software projects. Experience with several programming languages, including Java, JavaScript, C++, or Python, and software development tools. Strong skills in applied mathematics and algorithm design are required. Experience with distributed parallel computing or 3D visualization applications are a plus. Excellent interpersonal, written, and oral presentation skills are essential. Note, this position is reviewed annually on the basis of performance and can be renewed for a maximum of three years. Our Environment: The Structural Bioinformatics Group (http://bioinformatics.sdsc.eduhttp://bioinformatics.sdsc.edu/ http://bioinformatics.sdsc.eduhttp://bioinformatics.sdsc.edu/) at the San Diego Supercomputer Center (SDSC) (http://www.sdsc.eduhttp://www.sdsc.edu/ http://www.sdsc.eduhttp://www.sdsc.edu/) is involved in research and development activities centered around 3D structures of proteins and nucleic acids, the integration of structural data with other domains such as Medicine, Genomics, Biology, Drug Discovery, and the development of scalable solution to Big Data problems in Structural Bioinformatics. Our group leads the RCSB Protein Data Bank (PDB) west-coast operations. The RCSB PDB (http://www.rcsb.orghttp://www.rcsb.org/ http://www.rcsb.orghttp://www.rcsb.org/) represents the preeminent source of experimentally determined macromolecular structure information for research and teaching in biology, biological chemistry, and medicine. With over 300,000 unique users from over 160 countries around the world, the RCSB PDB is one of the leading worldwide Biological Databases. Our group is involved in the National Institutes of Health (NIH) Big Data to Knowledge (BD2K) initiative. As an Organized Research Unit of UC San Diego, SDSC is a world leader in data-intensive computing and cyber infrastructure, providing resources, services, and expertise to the national research community, including industry and academia. To apply, please send cover letter and resume to Dr. Peter Rose (pwr...@ucsd.edu mailto:pwr...@ucsd.edu). -- Peter Rose, Ph.D. Site Head, RCSB Protein Data Bank West (http://www.rcsb.orghttp://www.rcsb.org/ http://www.rcsb.orghttp://www.rcsb.org/) Principal Investigator, Structural Bioinformatics Laboratory (http://bioinformatics.sdsc.eduhttp://bioinformatics.sdsc.edu/ http://bioinformatics.sdsc.eduhttp://bioinformatics.sdsc.edu/) San Diego Supercomputer Center (http://www.sdsc.eduhttp://www.sdsc.edu/
Re: [ccp4bb] Interesting DNA contamination
Dear Stefan, just saw this after reading post: http://www.nature.com/nature/journal/v522/n7557/full/nature14559.html Best, Guenter Pramod, You already got good suggestions on how to handle DNA contamination in protein preparations. Let me point out briefly that you haven't demonstrated yet that your contamination is DNA. I had the same observation when purifying UvsX. A very persistent and strong contamination in all my preps at ~500kb. To test weather it was DNA or RNA I boiled the protein 30 minutes and incubated it with DNAase and RNAse and result was the same. I concluded it was neither RNA nor DNA and continued as if nothing had happened. This publication is reporting the same observation: Formosa and Alberts (1986) Purification and characterization of the T4 bacteriophage uvsX protein. J Biol Chem. 1986 May 5;261(13):6107-18. If you ever find out what it is that runs like 500kb DNA on Agarose, please let me know. S.
Re: [ccp4bb] Interesting DNA contamination
Hi Thanks all, I appreciate all the valuable inputs.. Piush.. I ll be trying Benzonase up next.. but since the DNA appears so secluded for DNAses, it makes me little skeptical as mg and DNAse already been there for ON dialysis. Tim if the DNA binds to the protein, wouldn't this affect the interpretation of the Agarose gel? I can send you the Ni-elution SDS pic where the GFP taged protein and DNA band appers at two different different and distinct sizes. 500 bases should result in a distinct shift during gel filtration. Do you observe this? Since I don't have free protein to compare with DNA-bound, m not able to see the shift of peak. While waiting for suggestions, you might set up crystallization trials just in case? Yea, just did that :) .. Tom Why not crystallize the whole complex? It would be more interesting than the protein alone- higher impact and all that. kept the trays, wish me good luck :) Dan 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. Yes, Its nice piece of info. Just one concern eats me, that is the longer wash that I observed some time with mem-proteins may lead to release of native bound lipids and causes deterioration. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase breathing) to disrupt the interaction. I ll be doing that provided it tolerate. 3) Combine salt with low concentrations of denaturants. Incorporated to optimization list 4) Try a couple of different restriction enzymes such as DpnI or FatI to see if you can break it into smaller fragments. If you can, you maybe able to clone into a cloning vector with compatable ends/blunt ligation to sequence and identify the region of host DNA that is causing the problem. Excellent input, ll be doing. ... paul Stepwise addition to 1% PEI (polyethylenimine) following cell lysis (before dialysis) should do the trick. You mean, membrane and protein extraction as well Ni-Aff in presence of ~1% PEI? ... Katharina Since your protein apparently binds ATP – do you know the active site? Maybe its worthwhile to do an active site mutant (or any other possible DNA binding mutant?) and to see if it makes any difference in DNA binding? Nice input, Working on it. Another question is, if the DNA you observe is somehow an homogeneous species. Yes, consistantly Otherwise, maybe there’s a way to somehow digest the DNA further, in presence of the protein, after purification to obtain an homogeneous species, than to get rid of the nucleases again and than to simply set up crystallization trays. To set up crystalliyation trials I would do anyways, in case you have enough protein. Kept Trays, :) Thanks again :) Pramod
Re: [ccp4bb] Interesting DNA contamination
That an entire 500bp would be protected from DNase seems very very strange - but very interesting if true! Could that band on the agarose gel be something else? Protein will stain a bit with ethidium as well as with coomassie. Did you add SDS before running the agarose gel or is it native (in which case the size of an intact protein-DNA complex is not directly related to the mobility, as others have noted)? How do the 280 and 260nm peaks in your UV spectrum compare to those expected for your protein (before adding ATP, and possibly expecting 1 ATP / molecule to come along for the ride during purification)? Good luck, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago pr...@uchicago.edumailto:pr...@uchicago.edu
Re: [ccp4bb] Interesting DNA contamination
Pramod, You already got good suggestions on how to handle DNA contamination in protein preparations. Let me point out briefly that you haven't demonstrated yet that your contamination is DNA. I had the same observation when purifying UvsX. A very persistent and strong contamination in all my preps at ~500kb. To test weather it was DNA or RNA I boiled the protein 30 minutes and incubated it with DNAase and RNAse and result was the same. I concluded it was neither RNA nor DNA and continued as if nothing had happened. This publication is reporting the same observation: Formosa and Alberts (1986) Purification and characterization of the T4 bacteriophage uvsX protein. J Biol Chem. 1986 May 5;261(13):6107-18. If you ever find out what it is that runs like 500kb DNA on Agarose, please let me know. S.
Re: [ccp4bb] Interesting DNA contamination
Correction, I meant to say 0.5kb, not 500kb sorry for that. S.
[ccp4bb] Interesting DNA contamination
Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
Re: [ccp4bb] Interesting DNA contamination
Dear Pramod Kumar, if the DNA binds to the protein, wouldn't this affect the interpretation of the Agarose gel? 500 bases should result in a distinct shift during gel filtration. Do you observe this? While waiting for suggestions, you might set up crystallisation trials just in case? Best wishes, Tim On 06/25/2015 11:23 PM, Pramod Kumar wrote: Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar -- -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] Interesting DNA contamination
pramod Do a spin after bacterial cell breaking at 8K to get rid of a nuclear pellet Also there is a nuclease preparation available called benzonase it is most effective when you also add magnesium in your buffer so in your homogenization buffer add magnesium and should avoid any EDTA Pius On Thu, Jun 25, 2015 at 2:23 PM, Pramod Kumar pramod...@gmail.com wrote: Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar -- P
Re: [ccp4bb] Interesting DNA contamination
Stepwise addition to 1% PEI (polyethylenimine) following cell lysis (before dialysis) should do the trick. --paul On 06/25/2015 05:23 PM, Pramod Kumar wrote Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
Re: [ccp4bb] Interesting DNA contamination
Several different approaches may help you to separate DNA from protein including; 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase breathing) to disrupt the interaction. 3) Combine salt with low concentrations of denaturants. 4) Try a couple of different restriction enzymes such as DpnI or FatI to see if you can break it into smaller fragments. If you can, you maybe able to clone into a cloning vector with compatable ends/blunt ligation to sequence and identify the region of host DNA that is causing the problem. Dan Daniel A. Bonsor PhD Institute of Human Virology, University of Maryland at Baltimore 725 W. Lombard Street N571 Baltimore MD 21201 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar [pramod...@gmail.com] Sent: Thursday, June 25, 2015 5:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Interesting DNA contamination Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
Re: [ccp4bb] Interesting DNA contamination
I think the more interesting questions are: should one want to disrupt such a tight interaction? Wouldn’t the structure of the protein bound to DNA be more interesting than the protein alone? Why not try to crystallize the complex and show how the protein binds? Sometimes you should just run with what Nature gives you. Cheers, tom From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bonsor, Daniel Sent: Friday, 26 June 2015 11:20 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Interesting DNA contamination Several different approaches may help you to separate DNA from protein including; 1) When bound to the nickel column, wash the protein/beads with 1-2M NaCl or 1-2M KCl for a few hours to overnight. Increase ionic strength should disrupt protein-DNA interactions. 2) Use a low concentration of urea (1.5-2M) or guanidine hydrochloride (1M) to partial unfold (increase breathing) to disrupt the interaction. 3) Combine salt with low concentrations of denaturants. 4) Try a couple of different restriction enzymes such as DpnI or FatI to see if you can break it into smaller fragments. If you can, you maybe able to clone into a cloning vector with compatable ends/blunt ligation to sequence and identify the region of host DNA that is causing the problem. Dan Daniel A. Bonsor PhD Institute of Human Virology, University of Maryland at Baltimore 725 W. Lombard Street N571 Baltimore MD 21201 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pramod Kumar [pramod...@gmail.com] Sent: Thursday, June 25, 2015 5:23 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Interesting DNA contamination Dear all Sorry for off topic and lengthy post, but I came across a very unique DNA contamination during one membrane protein purification (a microbial external environment sensor/response protein) Already done * DNAse used as stranded protocol during cell break. * Membrane extraction to Ni-Affinity and Ovenight TEV dig done with 0.5M NaCl * Fluorescence size exclusion done with 0.1M NaCl * Well stable peak and pure protein profile observed * But final purified protein inherently contains specific 0.5 Kb DNA stretch visible through out purification (observed by running Agarose gel of protien sample @ each step) Approach failed * Buffer switched from HEPES to Na-PO4 * O.N. dialysis in presence of DNAse * Using Heparin Column purification between Ni-Aff and SE (failed and protein starts deterioration) * Since protein binds ATP (ATP and MgCl2 added after O.N. Dialysis/digestion) Now... I need help for * How to get rid of DNA without loosing active protein? * What are best lipids to dope as -ve DNA replacement? * Since protein is pure and ample, how likely I can get crystal hits with such big DNA attached? * What longest possible DNA can be crystallize/ed with protein? * How exclusive are some mem-spanign-proteins to provide anchorage of prokaryotic genomic stretch? Thanks in advance :) Pramod Kumar
