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
