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 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 > -- Wangsa Tirta Ismaya ===================================== Josef-Israelstraat 66, 9718 GN Groningen The Netherlands
