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: [email protected] 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 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: [email protected] > 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 ************************************** 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] **************************************
