Use of the NanoDrop spectrophotometer

Open the software of the NanoDrop by double clicking at the icon "ND-1000 V.3.2.1" on the desktop.

To measure the protein concentration at 280 nm press the "Protein A280" button. The programme will ask to place 2 μl of deionized water (dH₂O) on the lower sample pedestal and then press OK.

• In "Sample type" box choose "1 Abs = 1 mg/ml". (If you are measuring the concentration of BSA you can choose BSA). There is also a possibility to enter the molar or mass extinction coefficient of your protein by choosing "Other protein (E & MW)" and "Other protein (E 1%)" respectively in order to calculate the concentration automatically from the absorption. Please note that ExPASy gives a value corresponding to 0.1%
• Wipe the water from BOTH upper and lower sample pedestals with a paper tissue and place 4 μl buffer on the lower one. Press "Blank".
• Wipe away with a paper tissue and put 4 μl of the protein sample on the lower pedestal. Press "Measure". You do NOT need to dilute the protein sample as in normal spectrophotometers. Usually concentrations up to 50 mg/ml can be handled without dilution.
• If you have chosen as sample type "1 Abs = 1 mg/ml" then in the lower box "mg/ml" box you will see the absorption of the protein at 280 nm. Keep in mind that even though Nanodrop's path length is 1 mm it transforms it to the absorption you would get in a normal 10 mm path length spectrophotometer. (If you have entered an extinction coefficient as described before it will give you directly the concentration).
• Wipe both pedestals (especially for proteins it is good to do it as soon as you get the measurement to avoid evaporation of the solvent and sticking of the protein on the pedestals) and put your next buffer or sample. It is possible to measure multiple samples with the same buffer by blanking only once though it is suggested to blank inbetween to ensure that the pedestals remain clean. If you think they are dirty you can also put some water on them or wipe with a wet paper tissue.

If you want a wavelength other than 280 nm or a wavelength scan then you can select "UV-Vis" from the main menu. You can perform a wavelength scan from 220 nm to 750 nm.

• The handling, blanking and measuring is as above.
• In the output you will get the spectrum from 220 nm to 750 nm and you can move the two cursors to the wavelength of your choice or just type it in the two "λ₁" and "λ₂" boxes. Keep in mind though that these are 1 mm path length absorbances and if you want to transform it to the equivalent of a normal spectrophotometer you have to multiply by 10.

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