Re: [ccp4bb] Determining concentration of membrane protein
Hi Everyone, Thank you so much for your additional tips about various kits. My protein is tagged and when I cleave off the tag (a step that needs reducing agent), I will unfortunately have both detergent and reducing agent in my protein buffer. Nicolas, thanks for your word of caution. I can't therefore use Pierce RAC BCA kit but it looks like I should be able to use the 660 nm kit recommended by Ho (Thanks very much, Ho!). By extension, is it then the case that the BCA assay is not recommended when both detergent and reducing agent are present or is that just a peculiarity of the Pierce RAC BCA kit? Thanks very much to everyone who responded! Raji On Fri, Feb 14, 2014 at 9:49 AM, Patrick Loll pat.l...@drexel.edu wrote: No, because Bradford is based on the increase in absorbance when the dye moves from a hydrophilic environment to a hydrophobic one (like the protein interior, or like the interior of a micelle). When detergents are present in excess of their CMC, the change in absorbance from partitioning into the micelles is generally large compared to any signal due to protein binding; plus preparing a perfectly matched blank solution is challenging when dealing with protein-detergent solutions. I second Michael's recommendation--BCA works well. On 14 Feb 2014, at 1:45 AM, Niks wrote: Dear All, May be a stupid question. But if we take buffer with detergent as control (Blank), would not the difference in ODs using any of the methods used e.g. Bradford assay, gives protein concentration? Regards Nishant On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.eduwrote: Your basic choices for protein assays are: 1. Alkaline copper methods (e.g., Biuret and micro-biuret) 2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays) 3. Hydrophobic dye methods (e.g. Bradford) 4. UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] What really happens in XDSCONV?
Dear Kay, Concerning usage of programs, everybody has his/her preferences, but what could be simpler than a 2-liner XDSCONV.INP like INPUT_FILE=XDS_ASCII.HKL OUTPUT_FILE=temp.hkl CCP4 ! or CCP4_F or CCP4_I or SHELX or CNS and then running XDSCONV by running xdsconv? At least there's not much room for mistakes. Exactly! I beg to disagree, and this was the point of my question. The output of XDSCONV literally says that 190093 reflections are read, and [of those, my interpretation] 44047 are accepted. I may be a pedant but I can't read that output in any other way. To me it looks like it is reading only the reflections that already fall into the asymmetric unit and is ignoring all the others. So if XDSCONV is really doing what it is supposed to, I would suggest rephrasing that output line. good point about the rephrasing. I'll see to making the wording consistent between XDSCONV and XDS. Thanks for that. But irrespective of the wording, it does take all observations into account when calculating the intensity (and amplitude) of the unique reflections (as it should - ignoring reflections would not make sense, and would produce significantly worse data). Great, this was just the reassurance I was looking for. However, it does so not by calculating the geometric mean (which you seem to assume), but by calculating the weighted mean. Weighting is done with the variances, and here it also does not differ from (c)truncate or other programs. Sorry, that was a typo born of thinking I could quote the manual from memory. But yes, pedantry aside, I will start using pointless in my csh pipelines. please report back whether that changes (or even improves) your results! It is always very good when people compare programs in a meaningful way, but from my own experience I can say that meaningful comparisons are sometimes not entirely straightforward to get right. (for the German-speaking: wer misst, misst Mist!) Probably I will still be in too much of a hurry each time to make a meaningful and thorough comparison, but if time allows I will compare XDSCONV with pointless/scala. /Derek
Re: [ccp4bb] identifying protein crystals via visible light only?
Hi Richard, Do you require visible light only? In any case, i remember a neat paper describing using Hoffman modulation, as well as polarisation and phase contrast. Pure analog computing ;) What stroke me then (and now) is that the standard microscopy kit people use does not use the variety of optical tricks available (bar the wavelength doubling/halving trick some are using). Phase contrast, for instance, is such a simple add on to microscopes - a hole with a patch in the centre. Check that: J. Appl. Cryst. (2003). 36, 1295-1296 [ doi:10.1107/S0021889803013724 ] Microscope detection options for colorless protein crystals grown in lipidic cubic phases P. Nollert Cheers, Jose'
Re: [ccp4bb] What really happens in XDSCONV?
Hi Derek I strongly recommend comparing with pointless/aimless, *not* pointless/ scala (if you have the time!) Scala is obsolete and was superseded by Aimless several years ago (I think 2010, without checking...). I find that Aimless is not only much faster than Scala, but also scales better. On 16 Feb 2014, at 19:26, Derek Logan wrote: Probably I will still be in too much of a hurry each time to make a meaningful and thorough comparison, but if time allows I will compare XDSCONV with pointless/scala. /Derek Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH
[ccp4bb] Introducing: XDSIT Automated diffraction data processing based on processing by XDS
Dear all, I would like to introduce XDSIT, a command-line/GUI/database tool for automated processing of single or multiple diffraction datasets based on processing by XDS. http://michaelkrug.reussmedia.de/ Best wishes Michael Krug
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Hi Raji, I have no experience with membrane proteins, but I have used SUMO tags frequently. Unlike other proteases that cleave at a specific site (thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for cleavage. So if only about 50% of your protein is cleaved, this may indicate that about 50% of your protein is misfolded. You may just try to take the cleaved protein and use it and forget about recovering the uncleaved portion. While your yield will obviously be substantially reduced, you only really want correctly folded protein for structural or functional studies, and the inability of Ulp1 to cleave the SUMO tag could serve as means of removing misfolded protein from your sample. Matt On Sun, Feb 16, 2014 at 9:58 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
By the way, I have an unrelated question. In the crystal structures containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19 residues are absent. I am curious whether people tried a SUMO tag with these residues deleted. I am using the vector from invitrogen which seems to have the full-length ySUMO. Thank you, Chen On Sun, Feb 16, 2014 at 10:38 PM, Matthew Bratkowski mab...@cornell.eduwrote: Hi Raji, I have no experience with membrane proteins, but I have used SUMO tags frequently. Unlike other proteases that cleave at a specific site (thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for cleavage. So if only about 50% of your protein is cleaved, this may indicate that about 50% of your protein is misfolded. You may just try to take the cleaved protein and use it and forget about recovering the uncleaved portion. While your yield will obviously be substantially reduced, you only really want correctly folded protein for structural or functional studies, and the inability of Ulp1 to cleave the SUMO tag could serve as means of removing misfolded protein from your sample. Matt On Sun, Feb 16, 2014 at 9:58 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Sorry for my typo, it is Ulp1-SMT3 complex... On Sun, Feb 16, 2014 at 10:54 PM, Chen Zhao c.z...@yale.edu wrote: By the way, I have an unrelated question. In the crystal structures containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19 residues are absent. I am curious whether people tried a SUMO tag with these residues deleted. I am using the vector from invitrogen which seems to have the full-length ySUMO. Thank you, Chen On Sun, Feb 16, 2014 at 10:38 PM, Matthew Bratkowski mab...@cornell.eduwrote: Hi Raji, I have no experience with membrane proteins, but I have used SUMO tags frequently. Unlike other proteases that cleave at a specific site (thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for cleavage. So if only about 50% of your protein is cleaved, this may indicate that about 50% of your protein is misfolded. You may just try to take the cleaved protein and use it and forget about recovering the uncleaved portion. While your yield will obviously be substantially reduced, you only really want correctly folded protein for structural or functional studies, and the inability of Ulp1 to cleave the SUMO tag could serve as means of removing misfolded protein from your sample. Matt On Sun, Feb 16, 2014 at 9:58 AM, Raji Edayathumangalam r...@brandeis.edu wrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
