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
Re: [ccp4bb] Determining concentration of membrane protein
Hi Raji, i the kit i used for this purpose was from Pierce it's call Protein BCA RAC assay. BCA : for the colorimetric parts, en ad the RAC is for Reducing agent compatibility. This RAC is also efficient with detergent. (according my remember) But you if you use at the same time detergent and Reducing Agent, it's inefficient. http://www.piercenet.com/product/bca-protein-assay-reducing-agent-compatible If you use this one, it could be costless to buy the microplate oriented kit. (you can use also without microplate and you have more experiment in the same box) Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ho Leung Ng [h...@hawaii.edu] Envoyé : vendredi 14 février 2014 01:58 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein Hi Raji, There are also some proprietary stains such as the 660 nm (can't they think of a better product name?) stain from Pierce that are detergent compatible. I used this briefly with success when comparing against Abs 280 nm. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edumailto:h...@hawaii.edu Date:Thu, 13 Feb 2014 10:06:12 -0500 From:Raji Edayathumangalam r...@brandeis.edumailto:r...@brandeis.edu Subject: Determining concentration of membrane protein 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
Re: [ccp4bb] Determining concentration of membrane protein
The problem is that if you put detergent or reducing agent in Bradford or BCA, the reaction is complet also without protein. You can't determine the color gradient because every tubes are blue or purple. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Niks [nik...@gmail.com] Envoyé : vendredi 14 février 2014 07:45 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein 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.edumailto:rrowl...@colgate.edu wrote: 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.edumailto: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
Re: [ccp4bb] Determining concentration of membrane protein
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.edu wrote: Your basic choices for protein assays are: Alkaline copper methods (e.g., Biuret and micro-biuret) alkaline copper + molybdate methods (e.g., Lowry, BCA assays) Hydrophobic dye methods (e.g. Bradford) 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
[ccp4bb] Determining concentration of membrane protein
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
Re: [ccp4bb] Determining concentration of membrane protein
Roger, While I agree with your list, the BCA assay does not use molybdate (as we make it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and cupric sulfate pentahydrate). For membrane proteins, I prefer the BCA assay until the protein is pure enough to use A280. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Feb 13, 2014, at 10:39 AM, Roger Rowlett rrowl...@colgate.edu wrote: Your basic choices for protein assays are: Alkaline copper methods (e.g., Biuret and micro-biuret) alkaline copper + molybdate methods (e.g., Lowry, BCA assays) Hydrophobic dye methods (e.g. Bradford) 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
Re: [ccp4bb] Determining concentration of membrane protein
Hi Everyone, Thanks very much for your helpful responses and suggestions. I will use the BCA assay. Cheers, Raji On Thu, Feb 13, 2014 at 11:27 AM, R. M. Garavito rmgarav...@gmail.comwrote: Roger, While I agree with your list, the BCA assay does not use molybdate (as we make it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and cupric sulfate pentahydrate). For membrane proteins, I prefer the BCA assay until the protein is pure enough to use A280. Cheers, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@gmail.com* ** On Feb 13, 2014, at 10:39 AM, Roger Rowlett rrowl...@colgate.edu wrote: 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 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Determining concentration of membrane protein
Hi Raji, There are also some proprietary stains such as the 660 nm (can't they think of a better product name?) stain from Pierce that are detergent compatible. I used this briefly with success when comparing against Abs 280 nm. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu Date:Thu, 13 Feb 2014 10:06:12 -0500 From:Raji Edayathumangalam r...@brandeis.edu Subject: Determining concentration of membrane protein 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
Re: [ccp4bb] Determining concentration of membrane protein
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.edu wrote: 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
