For accurate absorbance measurements we have been using a 10 ul "tunnel" cuvette for some time. It is still 1 cm pathlength, fits into standard specs, one can manage even with 8 ul of a sample fed into the channel, which can be rescued ~ 80%, no evaporation issues, the cuvette has to be kept clean!
Jan D. On Fri, Jun 17, 2011 at 3:36 AM, Richard Edward Gillilan <r...@cornell.edu> wrote: > > Hi Chelsy, yes we had a lot of trouble with the nanoview during that run. > Even after going through the calibration procedure with the special fluid > provided, we still had inconsistent results even on standards. Finally, I > carefully cleaned the return light path of the instrument (a separate set of > holes towards the back of the glass plates). This seemed to fix the problem. > The manual warns that users should always wipe from back to front when > cleaning out sample. The instrument also seems sensitive to how well the > drop is formed on the glass plate. Once the hydrophobic coat is worn or > dirty, the drops spread and give poor results, so maybe detergent was also > an issue. So these machines can be fussy we are learning. > Richard Gillilan > MacCHESS > > On Jun 16, 2011, at 9:03 PM, Chelsy Prince wrote: > > Hi everyone, > > I am working with a membrane protein and normally measure my protein > concentration by diluting and then reading OD280 (1cm pathlength). I have > found this to be very consistent, if a bit time consuming. > > At the syncatron, I had to use a Nanodrop for the first time to check some > SAXS dilutions I made on site and it was all over the map. We tested each > sample 8-10 times and saw large variations (i.e. 2.5-4.1mg/mL) on a single > sample. I used the Nanodrop to measure OD280 and calibrated it with the same > extinction coefficient I normally use. > > Since I have dodecyl-maltoside in all of my solutions (which is the cause of > most of my problems), I was wondering if it was also the culprit here. > The detergent is probably lowering the surface tension of the drops. > > It is possible that I was just in-experienced, but I had multiple people > from the facility helping me and we all had the same issue with my samples. > In addition, the Nanodrop would regularly complain about inconsistency > between the two readings and wouldn’t accept readings/blanks at all. > > Is it me or the membrane protein? And is there anything that I could do to > improve the readings the next time that I need to use it? > > Thanks, > Chelsy > > -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 390 Fax: +420 296 809 410
