An important step in protein characterization is determining the concentration of a sample. The concentration of a protein sample provides a guideline for many experimental protocols, such as western blots, crystallization trials and kinetic assays. Two commonly used methods for determining protein concentration include the Bradford assay, which measures dye binding to arginine and aromatic residues, and the Bicinchoninic Acid (BCA) method, which measures reduction of copper ions by the protein.
Bradford Assay
Step 1
Dilute the Bradford dye with distilled water according to the manufacturer's instructions.
Step 2
Filter the dye solution using gravity filtration. This solution can then be used for 2 weeks.
Step 3
Prepare standards of known protein concentrations by adding 20 microliters (ul) of bovine serum albumin (BSA), in concentrations ranging from 0 to a high concentration of your choice, to separate test tubes. Add 5 ml of the diluted Bradford dye to each test tube.
Step 4
Add 20 ul of each protein sample you wish to know the concentration of to separate test tubes and add 5 ml of Bradford dye to each.
Step 5
Invert all tubes and allow them to sit at room temperature for 5 minutes. Take care to keep the incubation times consistent between samples, as the absorbance will increase with time.
Step 6
Set spectrophotometer to 595 nanometers (nm). Transfer 1 ml of the 0 concentration standard to a plastic cuvette and use it to blank the spectrophotometer.
Step 7
Measure the absorbance of the remaining standards individually at 595 nm and make a standard curve by plotting the absorbance value at 595 nm against the known concentration of BSA.
Step 8
Measure the absorbance of your protein samples of unknown concentration. Use the standard curve to determine the concentration of your protein samples based on the absorbance values of the standards.
Bicinchoninic Acid (BCA) Assay
Step 1
Prepare a working stock of dye by mixing Reagent A with Reagent B per the manufacturer's instructions. This working solution can be used for a week.
Step 2
Prepare standards by adding 200 ul of the BCA working solution to Eppendorf tubes containing 10 ul of BSA, in concentrations ranging from 0 to a high concentration of your choice. Be sure to make at least six different standards for an accurate standard curve.
Step 3
Prepare samples of unknown protein by adding 10 ul of each sample to 200 ul of BCA working solution in separate Eppendorf tubes.
Step 4
Vortex standards and samples and place at 37 degrees Celsius for 30 minutes. Cool to room temperature.
Step 5
Blank the spectrophotometer at 562 nm using the 0 concentration standard. Prepare a standard curve by reading the absorbances of the protein standards and plotting absorbance vs. concentration.
Step 6
Read the absorbance of all unknown protein samples and use the standard curve to estimate the concentration.
Things You'll Need
- Protein sample
- Bovine serum albumin
- Bradford assay reagent
- Bicinchoninic acid assay reagents A and B
- Spectrophotometer
- Eppendorf tubes
- Test tubes
- Plastic cuvettes
- Water bath
- Pipettes with volumes ranging 10 ul to 1 ml
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