[ccp4bb] Assemble Protein-DNA complex
Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei Huang, if you are lucky you can form the complex as crystal in the drop (I assume you want to crystallise the protein-DNA complex): set up drops at high salt concentration (as low as possible to keep the protein in solution at reasonable concentration) in the presence of DNA. Prepare the buffer the same way as the protein buffer, but with reduced salt concentration (btw, you can also try divalent ions, eg. MgCl2). This way water evaporates into the drop, diluting the salt concentration. - From your discription the solubility of the protein drops faster than its concentration, hence it should precipitate. And, as I said, if you are lucky, it does so as a crystal in complex with the DNA. Best, Tim On 11/09/2012 05:14 PM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 nvrZMfMmeekAauJ26jy6jbE= =nbV+ -END PGP SIGNATURE-
Re: [ccp4bb] Assemble Protein-DNA complex
Dear Wei, If i understand your different experiment, you try to obtain your protein DNA complex at different salt concentration with different method to reach the final concentration. I read that you try 150 mM KCl + 150 mM NaCl as high concentration salt, it result in 300 mM cations and 300 mM Cl. To my mind, and according your experiment it's too high. But when you try low concentration you dialized the protein alone in 30 and 100 mM salt concentration. In my opinion the best way, is to dialize the protein mixed with the DNA. Because the protein will probably be stablize by DNA. If the protein meet slowly DNA at the same time the salt concentration decrease, you minimize the precipitation (it's your third experiment in fact). You have not precised the final salt concentration with this method. If you try 30 mM, maybe it's too low, i suggest 100mM Salt. I remember paper about nucleosome where author explain solution with multiples steps dializis. It's more gradual and it could help if you want reach very low salt concentration. One other idea, is to play with the pH, because of the protein's pI. In function of the pI you have to try different pH for the complex preraration. You can optimise the protein DNA interaction through the protein charge. HtH Nicolas Le 09/11/12 17:14, Wei Huang a écrit : Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
This sounds like a job for ammonium acetate. Use it as your salt. Purify your complex in it and then set up drops where they wells have the amount of ammonium acetate needed to keep your protein stable and the wells have none, or a range of concentrations. The ammonium acetate will equilibrate by vapor diffusion, lowering the concentration in the drop and causing your complex to come out of solution. James On Nov 9, 2012, at 9:14 AM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
I meant where the drops have the concentration of ammonium acetate needed. James On Nov 9, 2012, at 10:06 AM, James Stroud wrote: This sounds like a job for ammonium acetate. Use it as your salt. Purify your complex in it and then set up drops where they wells have the amount of ammonium acetate needed to keep your protein stable and the wells have none, or a range of concentrations. The ammonium acetate will equilibrate by vapor diffusion, lowering the concentration in the drop and causing your complex to come out of solution. James On Nov 9, 2012, at 9:14 AM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, -- Wei Huang, PhD Postdoctoral Associate Center for Proteomics and Bioinformatics Case Western Reserve University Cleveland, OH 44106
Re: [ccp4bb] Assemble Protein-DNA complex
It is not very surprising that the affinity gets higher with lower salt, right? Why dont you measure it under _physiological_ salt concentration? (or i assume maybe you did?) and of course its not as high affinity due to screening (but physiological conditions) of the electrostatic interactions. If the complex doesnt stay together in ca. 150 mM salt (e.g in TBS) in gel filtration (which you dont say if you tried) then why not just try mixing it in the drop and screen, this is what you would do if you cant purify the complex. and try different rations. There are texts on preparation of DNA complexes in case you dont have advice for it. Tommi On Nov 9, 2012, at 6:50 PM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei Huang, if you are lucky you can form the complex as crystal in the drop (I assume you want to crystallise the protein-DNA complex): set up drops at high salt concentration (as low as possible to keep the protein in solution at reasonable concentration) in the presence of DNA. Prepare the buffer the same way as the protein buffer, but with reduced salt concentration (btw, you can also try divalent ions, eg. MgCl2). This way water evaporates into the drop, diluting the salt concentration. - From your discription the solubility of the protein drops faster than its concentration, hence it should precipitate. And, as I said, if you are lucky, it does so as a crystal in complex with the DNA. Best, Tim On 11/09/2012 05:14 PM, Wei Huang wrote: Dear CCP4BBers, I have a problem in purifying protein-DNA complex for a protein that I am interested in. The purification of protein only has been optimized and I've get enough yield for what I need (10 mg/2.4 L growth). And I've measured DNA binding using Fluorescence Anisotropy. The results show that my protein has the tightest binding (Kd=8 nM) to the DNA at low salt condition (30 mM KCl) in 20 mM HEPES (pH 7.5). However, I came across several problems when I assemble protein-DNA complex in large scale. First, my protein is unstable at low salt condition. When I dialyzes my protein into low salt buffer (tried 30 mM and 100 mM KCl) for binding DNA, the protein precipitates. What I don't quite understand is that the DNA binding assay performed at low salt condition doesn't seem to be affected by this instability of protein. I guess it may be due to the assay was performed at very diluted protein concentration (in nM). Second, I can not purify protein-DNA complex at high salt condition with gel filtration column. Because of the first problem, I tried to assemble the complex at high salt condition (150 mM KCl, 150 mM NaCl). However, the elution profile shows no binding of DNA to my protein (no increase in the observation of protein peak and a large peak around expected position for DNA). This may be due to weaker binding at high salt as my DNA binding assay shows that the Kd under this buffer condition is ~1100 nM. Third, a lot of protein is lost during dialysis of protein-DNA complex into low salt condition. I tried add DNA directly into protein in high salt buffer, then dialyze very slowly against low salt buffer. However, I still lost quite a lot of protein due to precipitation. I was able to load some sample onto the gel filtration column with low salt running buffer. And I saw the shift of protein peak in the elution profile, also protein concentration measured by Bradford assay shows that the protein concentration is much less than that expected from uv trace, suggesting the contribution to the absorbance from DNA. But the yield is very low, less than 0.2 mg of protein is left and the complex seems to be unhappy when I concentrate it. So I can not get protein sample concentrated enough for my study. My previous experience with another DNA binding protein is much better. I purified it in high salt, dialyzed into low salt to binding DNA and finally purify with gel filtration column. However, the one I am currently working on seems to be very picky. If you have any suggestion regarding to my problems, I will be thankful. Best regards, - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQnTRFUxlJ7aRr7hoRAvscAJ9Bm/3ix2ln69wZz74LWwaPMdBhFQCfS9C6 nvrZMfMmeekAauJ26jy6jbE= =nbV+ -END PGP SIGNATURE- Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi http://www.biocenter.helsinki.fi/bi/kajander/
Re: [ccp4bb] Assemble Protein-DNA complex
On 11/09/12 15:43, Tommi Kajander wrote: It is not very surprising that the affinity gets higher with lower salt, right? Not at all. I wanted to ask if their assay could distinguish between specific binding and nonspecific binding, but I decided not to sidetrack the discussion. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
