Protein concentration

Measuring protein concentration using NanoDrop

Both Rutger's University and University of Toronto use the UV absorbance at 280 nm to determine protein concentrations.

Absorbance by proteins is primarily due to the aromatic rings of Phe, Tyr, and Trp residues.

Extinction coefficients are estimated using the ExPASy ProtParam tool (a description of the equations used for this calculations can be found here )

Procedure (used at Rutger's University)

concentration (M) = Absorbance at 280 nm / ( extinction coefficient * path length)

The extinction coefficent has the units M-1 cm-1.  The path length of most UV spectrophotometers is 1 cm.

With the NanoDrop instrument, the path length is not 1 cm, but the Absorbance at 280 nm is corrected as if the pathlength is 1cm.  So, the equation to use is just:

concentration (M) = Absorbance at 280 nm / extinction coefficient 

Typical protein concentration measurement protocol with NanoDrop-2000 with connected PC computer.

1. Before starting the software module, clean the sample surfaces with DI water to remove any dried sample that might be present.
2. Alternatively, you can clean the sample surfaces with a Kimwipe moistened with 70% ethanol.
3. Open the Nanodrop program and the appropriate module (e.g., Protein).
4. Wipe off the top and bottom sensors of the instrument with a Kimwipe. These are just the polished ends of fiber optic cable, so wiping is sufficient to prevent carryover.
5. Pipette 2 μL of DD water onto the sensor. Bring down the lever arm.
6. Follow the onscreen prompts to calibrate.
7. Wipe the sensors and pipette on 2 μL of the corresponding blank (Buffer solution your protein is in). Bring down the lever arm.
8. Follow the onscreen prompts to blank.
9. Wipe the sensors and pipette on 2 μL of your protein sample. Bring down the lever arm.
10. Click Measure and record the concentration measured.
11. In order to determine protein concentration, make note of the intensity at 280 nm.
12. To test multiple samples, just wipe the sensor in between measurements with a Kimwipe. Recalibration or re-blanking is not necessary.
13. Clean the sample surfaces once more after you are finished.

Procedure (University of Toronto)

Since july of 2009, we use thermofisher nanodrop1000 to measure the protein absorbance at 280 nm.

The molecular weight and theoretical extinction coefficient are calculated using expasy.