Proteins are one of the major molecular components of your entire body and can be found in your blood, bones, cells, tissues and organs. Proteins are also an essential component of your nutritional intake because your body uses them to build its own proteins. Measuring and calculating the concentration of protein in a solution can be done by utilizing the ultraviolet light absorbing qualities of proteins.
Step 1
Prepare your protein sample. Use your blender or homogenizer to blend or homogenize a protein sample from plant or animal tissue. You should prepare these samples in a buffered extract solution with all non-soluble material removed by centrifugation or filtration.
Step 2
Set up the spectrophotometer. Set your spectrophotometer to read your sample at the ultraviolet wavelength of 280 nanometers, or nm. To correct for background absorbance, fill a 1 cm path length quartz cuvette with the necessary amount of your unused buffered extract solution and take an absorbance reading at 280 nm. If the option on the spectrophotometer is available, set this absorbance reading as the background, or zero absorption value.
Step 3
Measure your protein sample. Add a small amount of your clarified protein sample to the extract buffer in the cuvette that you used to measure background absorbance. Mix the sample well prior to reading and recording the absorbance at 280 nm in the spectrophotometer.
Step 4
Calculate your protein concentration. Subtract the background absorbance reading from the absorption value you recorded for your protein sample. In the generic calculation, the concentration of protein, in milligrams per milliliter, is equal to the absorption of ultraviolet light at 280 nm, divided by the length of the light path in centimeters. In this scenario, the path length is 1 cm, so the concentration of protein in milligrams per milliliter is equal to the absorption reading at 280 nm, divided by 1 cm.
Tips and Warnings
- Repeated measurements improve accuracy. After you have made one absorbance reading, clean out the cuvette and make additional measurements using the same process. This is only a general method for determining protein concentration. More detailed methods that would improve your accuracy would utilize standard curves that are generated using dilutions of known protein concentrations.
- Contamination with other molecules may affect your spectrophotometer readings. A common contaminant in crude protein extracts is the nucleic acids that make up DNA. These molecules also absorb some ultraviolet light at 280 nm.
Things You'll Need
- Protein sample
- Buffered extraction solution
- Homogenizer or blender
- Centrifuge or filter
- Ultraviolet spectrophotometer
- One-centimeter path-length quartz cuvette
- Calculator
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