Measurement

Overview

Most of the time, when we need to measure the concentration of protein in a solution, we would use a "microplate reader" to obtain precise values. However, for most high school teams, their laboratory setups rarely provide access to such advanced equipment. Therefore, our team has designed this relatively accurate and low-cost protein concentration measurement method, hoping it will not only serve our own project but also help more iGEM teams conduct their experiments smoothly.

The relatively accurate and low-cost protein concentration measurement method

Principle

This experiment is based on the principle that proteins exhibit characteristic absorbance at 280 nm wavelength, primarily due to tryptophan and tyrosine residues. By measuring the absorbance of standard proteins of known concentrations to establish a concentration-absorbance standard curve, the concentration of an unknown sample can be accurately determined from its absorbance value using this curve. This method is designed for situations with limited laboratory configuration, offering the advantages of speed, low cost, and minimal sample consumption.

Preparation

Standard Protein Stock Solution: Prepare a series of standard solutions of known concentrations using BSA(Bovine Serum Albumin) dissolved in the same buffer as the sample (e.g 10mM Tris-HCl, pH 9.0);

Blank Control Solution: Use the exact same buffer or solution used to dissolve the protein samples (e.g., if the sample contains imidazole, salts, etc., these must be added in equal amounts to the blank solution);

Instruments and Consumables: Nanodrop spectrophotometer, micropipettes and lens cleaning paper.

Procedure

Step1: Instrument Preparation and Calibration

  1. Using a micropipette, apply 1-2 μL of ultrapure water (ddH₂O) to both the upper and lower measurement pedestals of the Nanodrop, then close the measurement arm to rinse. Gently blot dry with a lens cleaning paper.
  2. Pipette 2 μL of the blank control solution onto the lower pedestal and close the measurement arm. Perform the "Blank" or "Blank Correction" operation in the instrument software. This step subtracts the buffer background, ensuring an accurate measurement baseline.

Step2: Standard Curve Measurement

  1. After cleaning the pedestals, sequentially pipette 2 μL of each different concentration BSA standard onto the pedestal for measurement. Critical: Ensure the pedestals are clean and dry before each sample application.
  2. Record the absorbance value at 280 nm (A280) for each standard. Also, note the identification number and known concentration of each standard in the lab notebook.
  3. If the reading for any standard falls outside the instrument's linear range, perform a precise dilution using the blank control solution, remeasure, and be sure to record the dilution factor.

Step3: Sample Measurement

  1. Clean the pedestals, then pipette 2 μL of the protein sample to be measured onto the pedestal for measurement.
  2. To minimize random error, perform at least three independent measurements for the same sample.
  3. Record the sample identification number, the A280 absorbance value for each measurement, and the sample's dilution factor (if applicable).

Step4: Data Processing and Analysis

  1. Create a scatter plot with standard protein concentration on the X-axis and the corresponding A280 absorbance value on the Y-axis (called plot standard curve).
  2. To fit the data to a linear function Y = aX + b (where Y is absorbance and X is concentration).
  3. Substitute the average absorbance value of the test sample into the fitted equation to calculate its concentration. If the sample was diluted, multiply the result by the corresponding dilution factor to obtain the original concentration.
  4. Calculate the average and standard deviation of the sample concentrations from all valid readings to evaluate the repeatability and reliability of the results.

Matters need attention

  1. The measurement volume for all standards and samples must remain strictly consistent throughout the procedure (2 μL in this protocol). This is fundamental for data comparability.
  2. The solvent background of the blank control must perfectly match that of the standards/samples. This is a critical prerequisite for experimental accuracy.
  3. Taking multiple readings and averaging the results for key samples is a simple and effective method to ensure stable and reliable data.

experimental data

Standard protein:

protein concentration(mg/ml) 1.5 1 0.75 0.5 0.25 0.125 0
A280 0.858 0.571 0.423 0.285 0.132 0.078 0.016

Samples:

number of samples alpha-3_1 alpha-3_2 alpha-3_3 alpha-3_4 alpha-3_5 r-5_1 r-5_2 r-5_3
A280 0.73 0.61 0.84 0.52 0.67 0.47 0.29 0.38
Calculated concentration(mg/ml) 1.269 1.058 1.464 0.898 1.163 0.810 0.492 0.651

Fitted Equation: Y = 0.566X + 0.0114

R² Value: 0.9991

(The R² value > 0.99 indicates an excellent linear relationship between protein concentration and absorbance.)

Conclusion:

Alpha-3 Samples:

  • Mean Concentration: 1.170 mg/mL
  • Standard Deviation: 0.218 mg/mL

Gamma-5 Samples:

  • Mean Concentration: 0.651 mg/mL
  • Standard Deviation: 0.159 mg/mL