Re: [ccp4bb] suggestions for UV spectrometer
nanodrop system is wonderful. it helps very much in solutions with high interference from detergents etc etc. i highly receommend nanodrop specs PSP On Thu, Dec 4, 2008 at 10:16 AM, Tim Gruene [EMAIL PROTECTED] wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Pius S Padayatti Scientist, Polgenix, Inc. 11000 Cedar Ave, Suite 260 Cleveland, OH 44106 Phone: 216-658-4528 Fax: 216-658-4529
Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
I also have to come in defense of the nanodrop here. I have measured up to A280 = 98 and the curve is always reasonably smooth, spikes normally mean that bubbles have formed. And proper cleaning seems to be rubbing with a kimwipe three or four times after each drop. If the last user does not clean after the last measurement, then things will dry up in the pedestals and that can of course be a problem. Also bubbles do not form if you are careful. I always use 2uL, 1uL can be too little, and even with 20% detergent I get a column and no bubbles nearly every time. You have to pipet carefully and lower the lever slowly (think of a vinyl record here). For tricky samples, use 2.2uL instead. A bubble might still form occasionally, but looking at the spectrum will quickly tell you that something went wrong. Cheers, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Dec 5, 2008, at 7:00 PM, wangsa tirta ismaya wrote: Dear all, Thanks a lot for raising the issue with the not reproducibility of protein measurement with Nanodrop. We use the instrument as a workhorse in the lab. Indeed, recently I observed that the protein concentration suggested by Nanodrop is sometimes differ to the usual colorimetric measurement (Bradford method, measured with our Pharmacia's Ultrospec 2000 spectrometer). Since the cell in Nanodrop is very small, could it be due to the homogeneity of the sample in the cell? Also what I have observed, we have to be sure that the cell is cleaned properly before use. Another thing is, at high protein concentration I obtained noisy absorption curve at the peak (like a seismograf ) thus the protein concentration measured is doubtful, I have to dilute the sample to have good curve (thank God it requires only 2 mikroliter for a measurement). Well, I think nanodrop is a good, fast, and powerful instrument, however it would be better if we established a reference of our daily practice to a normal spectrometer measurement. cheers, Wangsa 2008/12/5 Martin Hallberg [EMAIL PROTECTED] Which brings us back to the Hellma TrayCell solution where you can, from the same spectrometer, have both the cuvette option and the quickness of the NanoDrop/NanoVue system. Anyone that can comment on the performance of the TrayCell from Hellma? Cheers, Martin On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote: Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a real 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There's also one quite strange thing about the nanodrop – they sell the calibration check solution (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it's off to some certain extend: But then you're stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration-option into the software, but at the moment the instrument tells you calibration failure and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them with locktite. Anyway, at least for our mid to high concentrated samples the nanodrop is not showing large fluctuations so we are happy with it. But everyone using a nanodrop should check it from time to time – as I found out that ours was off more than 20% at one day - which raised some trouble of course… Cheers, Gregor Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Filip Van Petegem Gesendet: Donnerstag, 4. Dezember 2008 22:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] suggestions for UV spectrometer I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard
Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
Getting even further off topic, we had a Nanodrop demo for trial shortly after hearing that a significant number of crystallization groups were using them. I won't go into the trial details, but I'd be interested in hearing from people (via email rather then the ccp4 postings) who did systematic trials in comparison to more conventional spectrometers; especially concerning the accuracy and reproducibility. The ease of use was very nice but we did not buy one. Thanks, Eddie. Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jose Antonio Cuesta-Seijo Sent: Monday, December 08, 2008 12:54 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer I also have to come in defense of the nanodrop here. I have measured up to A280 = 98 and the curve is always reasonably smooth, spikes normally mean that bubbles have formed. And proper cleaning seems to be rubbing with a kimwipe three or four times after each drop. If the last user does not clean after the last measurement, then things will dry up in the pedestals and that can of course be a problem. Also bubbles do not form if you are careful. I always use 2uL, 1uL can be too little, and even with 20% detergent I get a column and no bubbles nearly every time. You have to pipet carefully and lower the lever slowly (think of a vinyl record here). For tricky samples, use 2.2uL instead. A bubble might still form occasionally, but looking at the spectrum will quickly tell you that something went wrong. Cheers, Jose. ** Jose Antonio Cuesta-Seijo Cancer Genomics and Proteomics Ontario Cancer Institute, UHN MaRS TMDT Room 4-902M 101 College Street M5G 1L7 Toronto, ON, Canada Phone: (416)581-7544 Fax: (416)581-7562 email: [EMAIL PROTECTED] ** On Dec 5, 2008, at 7:00 PM, wangsa tirta ismaya wrote: Dear all, Thanks a lot for raising the issue with the not reproducibility of protein measurement with Nanodrop. We use the instrument as a workhorse in the lab. Indeed, recently I observed that the protein concentration suggested by Nanodrop is sometimes differ to the usual colorimetric measurement (Bradford method, measured with our Pharmacia's Ultrospec 2000 spectrometer). Since the cell in Nanodrop is very small, could it be due to the homogeneity of the sample in the cell? Also what I have observed, we have to be sure that the cell is cleaned properly before use. Another thing is, at high protein concentration I obtained noisy absorption curve at the peak (like a seismograf ) thus the protein concentration measured is doubtful, I have to dilute the sample to have good curve (thank God it requires only 2 mikroliter for a measurement). Well, I think nanodrop is a good, fast, and powerful instrument, however it would be better if we established a reference of our daily practice to a normal spectrometer measurement. cheers, Wangsa 2008/12/5 Martin Hallberg [EMAIL PROTECTED] Which brings us back to the Hellma TrayCell solution where you can, from the same spectrometer, have both the cuvette option and the quickness of the NanoDrop/NanoVue system. Anyone that can comment on the performance of the TrayCell from Hellma? Cheers, Martin On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote: Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a real 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There's also one quite strange thing about the nanodrop - they sell the calibration check solution (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it's off to some certain extend: But then you're stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration-option into the software, but at the moment the instrument tells you calibration failure and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them
Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
We have used Nanodrop for several years and found the readings are always accurate. The highest concentration we have measured is around 30 mg/ml. The differences between diluted and concentrated samples are within dilution error. Nanodrop spectra at low concentration are noisier. We actually prefer to have protein concentrations over 0.5 mg/ml. The key to get good Nanodrop reading is to have a nice liquid column. Take a look whiling waiting for the results. High concentration samples with detergent or glycerol may produce bubbles (giving an error message sometimes) or improperly formed liquid column (giving incorrect numbers). Use more samples (up to 5 ul) and gently push down the head will help the liquid column formation. 1 ul is not enough for most protein samples. If the reading is not good, wipe clean the surface and load a new sample. Repeated reading of same sample gave huge variation. The liquid column probably has difficulty to form properly after the first reading. We provide protein purification contract services and send proteins to many users around the globe. The proteins have been used for crystallography, ITC, DSC, Biacore, and activity assays. So far we havent heard any complain about our concentration measurement using Nanodrop. We also use Bradford to measure concentration if required by our customers or when concentration cant be measured by A280. The error range for Bradford is much wider and easily off by a factor of 2 (by repeating or comparing to Nanodrop). There are different brands of Bradford reagent. Each gives a different number. We once had a big discrepancy with a customer. It turns out the home-made BSA standard lot was off. After adjusting the BSA concentration with Nanodrop and using the same Bradford reagent and same curve fitting method, the concentrations agreed. The lesson leaned is to have good fresh standard solution. The color of Bradford changes over time, its better to use a plate reader than single sample spectrometer. Estimation by eye is just as good as a spectrometer:-) --Chun _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Juergen Bosch Sent: Saturday, December 06, 2008 12:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer Hi, really strange, I always dilute my protein when taking an absorption spectra. I try to adjust my expected concentration to a readout of ~ 0.2-0.3 OD280. And the Bradford is just as well as 'picking house numbers', depending on your protein, you can underestimate your protein concentration by a factor of 2. For my current approach I use a 100 µl volume cuvette and 2 µl of my concentrated protein (most of the times). The Structural Genomics lab (MSGPP) has a Nanodrop and I was also always happy with those results (but again I dilute my protein sample before taking a measurement) Jürgen On 5 Dec 2008, at 16:00, wangsa tirta ismaya wrote: Dear all, Thanks a lot for raising the issue with the not reproducibility of protein measurement with Nanodrop. We use the instrument as a workhorse in the lab. Indeed, recently I observed that the protein concentration suggested by Nanodrop is sometimes differ to the usual colorimetric measurement (Bradford method, measured with our Pharmacia's Ultrospec 2000 spectrometer). Since the cell in Nanodrop is very small, could it be due to the homogeneity of the sample in the cell? Also what I have observed, we have to be sure that the cell is cleaned properly before use. Another thing is, at high protein concentration I obtained noisy absorption curve at the peak (like a seismograf ) thus the protein concentration measured is doubtful, I have to dilute the sample to have good curve (thank God it requires only 2 mikroliter for a measurement). Well, I think nanodrop is a good, fast, and powerful instrument, however it would be better if we established a reference of our daily practice to a normal spectrometer measurement. cheers, Wangsa 2008/12/5 Martin Hallberg [EMAIL PROTECTED] Which brings us back to the Hellma TrayCell solution where you can, from the same spectrometer, have both the cuvette option and the quickness of the NanoDrop/NanoVue system. Anyone that can comment on the performance of the TrayCell from Hellma? Cheers, Martin On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote: Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a real 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There's also
[ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a real 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There's also one quite strange thing about the nanodrop - they sell the calibration check solution (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it's off to some certain extend: But then you're stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration-option into the software, but at the moment the instrument tells you calibration failure and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them with locktite. Anyway, at least for our mid to high concentrated samples the nanodrop is not showing large fluctuations so we are happy with it. But everyone using a nanodrop should check it from time to time - as I found out that ours was off more than 20% at one day - which raised some trouble of course. Cheers, Gregor Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Filip Van Petegem Gesendet: Donnerstag, 4. Dezember 2008 22:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] suggestions for UV spectrometer I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll [EMAIL PROTECTED] wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules shadowing one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] AW: [ccp4bb] suggestions for UV spectrometer
Which brings us back to the Hellma TrayCell solution where you can, from the same spectrometer, have both the cuvette option and the quickness of the NanoDrop/NanoVue system. Anyone that can comment on the performance of the TrayCell from Hellma? Cheers, Martin On Dec 5, 2008, at 9:06 AM, Gregor Witte wrote: Agree! I think for crystallographic use the nanodrop is perfectly okay to see if the protein is 5mg/ml or 30mg/ml. But in fact I also do not trust our instrument if it comes to more important issues like preparing solutions for titrations or assays. And due to the small pathlength I do not trust absorptions of small concentrated samples at all. I always prefer a “real” 2-beam spectrophotometer (monochromators) with a quarz-cuvette and a nice pathlength. Of course, you cannot reliably measure solutions exceeding Abs 1 or maybe 1.5 OD in a spectrophotometer with 1cm pathlength. There’s also one quite strange thing about the nanodrop – they sell the “calibration check solution” (which is some kind of yellow chromate-solution with known absorption), then you check your nanodrop with it and maybe find out that it’s off to some certain extend: But then you’re stuck(!), because you cannot calibrate it on your own. I guess it would be quite easy to integrate a calibration- option into the software, but at the moment the instrument tells you “calibration failure” and you have to call the service guys who then carry it home and calibrate it by turning of the two small screws at the top of it and then glue them with locktite. Anyway, at least for our mid to high concentrated samples the nanodrop is not showing large fluctuations so we are happy with it. But everyone using a nanodrop should check it from time to time – as I found out that ours was off more than 20% at one day - which raised some trouble of course… Cheers, Gregor Von: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Im Auftrag von Filip Van Petegem Gesendet: Donnerstag, 4. Dezember 2008 22:20 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] suggestions for UV spectrometer I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll [EMAIL PROTECTED] wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules shadowing one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/ . B. Martin Hallberg, PhD Assistant professor Department of Cell and Molecular Biology Medical Nobel Institute Karolinska Institutet Von Eulersv. 1 SE-171 77 Stockholm Sweden Fax: +46-8-339380
[ccp4bb] suggestions for UV spectrometer
Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
Dear Tim -- On 4 Dec 2008, at 15:16, Tim Gruene wrote: we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. I love the Nanodrop system... I know you said you don't want anything fancy, but I like to spend as little protein as possible when measuring its concentration, and I like it fast and reproducible. Plus, in my hands, the Nanodrop system give very reproducible results. Small problem... expensive... You can find infos there: http://www.nanodrop.com/nd-1000-overview.html HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] suggestions for UV spectrometer
I would also recommend the nanodrop. It takes a whole spectra every measurement and there is no need to dilute your sample. You can demo it for a week and try it out. James M. Vergis, Ph.D. University of Virginia Molecular Physiology and Biological Physics MKWEINR 360A Snyder Building 480 Ray C. Hunt Drive PO Box 800886 Charlottesville, VA 22908-0886 phone: 434-243-2730 FAX: 434-243-8271 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Tim Gruene Sent: Thursday, December 04, 2008 10:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] suggestions for UV spectrometer Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
We use a Beckman Coulter DU730. It has a small footprint if lab space is an issue. It will do single-wavelength, multi-wavelength, or take an entire spectrum in 0.5 nm steps if you desire. It comes with the standard 1 ml cuvette holder, but we also purchased the microcuvette accessory for volumes in the 100 uL range. We've been using the instrument for over a year now with no problems. Results for determining protein concentrations using the method of Pace et al * *Protein Sci.javascript:AL_get(this,%20'jour',%20'Protein%20Sci.');1995 Nov;4(11):2411-23 are very reproducible using this spec. On Thu, Dec 4, 2008 at 10:37 AM, James M. Vergis [EMAIL PROTECTED]wrote: I would also recommend the nanodrop. It takes a whole spectra every measurement and there is no need to dilute your sample. You can demo it for a week and try it out. James M. Vergis, Ph.D. University of Virginia Molecular Physiology and Biological Physics MKWEINR 360A Snyder Building 480 Ray C. Hunt Drive PO Box 800886 Charlottesville, VA 22908-0886 phone: 434-243-2730 FAX: 434-243-8271 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Tim Gruene Sent: Thursday, December 04, 2008 10:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] suggestions for UV spectrometer Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 [EMAIL PROTECTED] [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
Tim - I would recommend a spectrometer that records entire spectra, instead of one that takes readings at just 280 nm. Contributions from light scattering can be very strong and can give results that deviate from the true value by a factor of two or more. One cannot detect scattering without recording spectra. The most severe case we have had was someone who thought the protein concentration was 10 mg/mL (based on 280) when in reality (after subtraction of the scattering contribution) it was only 4 mg/mL. Lots of other, less severe cases as well. Hope that helps. Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 4, 2008, at 9:16 AM, Tim Gruene wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules shadowing one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
We too have a nano-drop. We really like it , but have not yet fully switched over. I agree with all the good things said about it , but here are the few times the nano drop falls short: 1) We still use the old spec ( 1 cm path length ) for things at a very low concentration , i.e for the monitoring free thiols in protein with ellman reagent , the absorbances are very low and give poor reproducibility on the nanodrop because of small path-length . Of course this can be overcome by using a lot of protein at a higher concentration and modifying the assay. 2) For very concentrated membrane protein samples which tend to have a large concentration of detergent . The reproducibility is not very good because of the high concentration of deteregent preventing a proper meniscus from forming. The solution to this is to dilute your sample so the dteergent concentration is manageable ( a few times the CMC instead of tens of times the CMC Other than for these issues we almost entirely use the nanodrop and would gladly recommend it Hari On Thu, Dec 4, 2008 at 1:27 PM, Michael Giffin [EMAIL PROTECTED] wrote: We also like the Nanodrop. Very fast, no cuvettes (breaking, washing, cleaning, uh nitric acid bath anyone?), and the .ndv data file is a delimited text file. Open in a text editor, copy and paste into a spreadsheet, and you have a convenient record of all of your stocks, including date, sample name, concentration, and full spectra. It is expensive, but so are good cuvettes. Mike Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758 On Thu, Dec 4, 2008 at 7:16 AM, Tim Gruene [EMAIL PROTECTED] wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] suggestions for UV spectrometer
Hi Tim, The Shimadzu UV2401PC comes at a reasonable price and has all features you might possible need in a Biochemistry lab. And if you like the option to use small sample volumes, I would suggest to by a TrayCell from Helma - you put it in a regular photometer, it guides the light through a microliter chamber and back into the photometer (it is essentially a cuvette with optics and a 1 or 5 microliter chamber). Costs a few hundred Euros . If you're sure you will never need regular cuvettes - go for Nanodrop. If you want more flexibility, Shimadzu plus TrayCell . Best Clemens --- Jun.-Prof. Dr. Clemens Steegborn Ruhr-University Bochum Physiological Chemistry, MA 2/141 Universitaetsstr. 150 44801 Bochum, Germany phone: ++49 234 32 27041 fax: ++49 234 32 14193 email: [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll [EMAIL PROTECTED] wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules shadowing one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED] -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
