Protein Quantification Assay

Protein quantification is a common technique in experimental biology, used to measure the concentration or total amount of protein in a sample. This measurement is very important for biochemistry, molecular biology, medical research, and other fields.

 

Analysis Workflow

1. Sample Preparation

Cell extract, tissue samples, serum, plasma, urine or other biological samples containing protein. Ensure the sample is clean and free of impurities.

 

2. Choose the Quantification Method

Choose the protein quantification method that suits your experimental needs.

 

3. Prepare a Standard Curve

Prepare a series of standard samples with known concentrations, you need a standard curve to determine protein concentration.

 

4. Sample Measurement

Mix the sample with the reagent in the quantification method, and perform the reaction following the steps required by the method. Measure and record the results using a spectrophotometer or other relevant instrument based on the color, absorbance, or other characteristics of the reaction product.

 

5. Calculate Protein Concentration

Calculate the protein concentration in the sample based on the standard curve or the formula of the quantification method.

 

Common Methods of Protein Quantification

1. Bradford Protein Assay

(1) Principle: This method is based on the color change that occurs when Coomassie Brilliant Blue G-250 dye binds to protein.

(2) Advantages: Simple operation, higher sensitivity.

(3) Disadvantages: Interference from detergents and other chemicals.

 

2. Lowry Method

(1) Principle: Based on the binding of copper ions with peptide bonds in protein under alkaline conditions and further reaction with Folin-Ciocalteu reagent to produce color change.

(2) Advantages: Suitable for lower concentration of protein.

(3) Disadvantages: The steps are more complicated than the Bradford method and are prone to interference from other substances.

 

3. BCA Method (Bicinchoninic Acid method)

(1) Principle: Similar to the Lowry method, but using BCA reagent, which is more sensitive to the reaction of copper ions and protein.

(2) Advantages: Suitable for the quantification of trace proteins, good stability.

(3) Disadvantages: Requires a longer incubation time.

 

4. Ultraviolet Absorption Spectroscopy Method

(1) Principle: The tryptophan and tyrosine in protein have specific absorption peaks under ultraviolet light.

(2) Advantages: Fast and does not require the addition of any reagents.

(3) Disadvantages: Sensitive to the absorption interference of other substances, requires a rather pure sample.

 

5. Protein Quantification Based on Mass Spectrometry

(1) Label-free Quantification

① Principle: Without using chemical labels, protein is quantified directly by analyzing the intensity or count of peptide ions.

② Advantages: Simplified operation, applicable to a wide range of sample types, no additional chemical modification required.

③ Disadvantages: High requirements for sample preparation and mass spectrometry operation quality, complex data processing and interpretation.

 

(2) iTRAQ/TMT

① Principle: Special chemical labels are used to label proteins in the sample. In mass spectrometry, the labels generate specific fragmentation ions, which are used for quantitative analysis.

② Advantages: Multiple samples can be analyzed simultaneously, increasing the throughput of the experiment.

③ Disadvantages: High cost, complex data processing, high requirements for experimental design and execution.

 

(3) Isotope Coded Affinity Tag (ICAT)

① Principle: Proteins or peptides are labeled with chemical tags containing different isotopes, and then relative abundance of proteins in different samples is compared using mass spectrometry.

② Advantages: Provides accurate relative protein quantity comparison, can be used for complex samples.

③ Disadvantages: Higher cost, complex operation, may not detect unlabeled proteins.

 

(4) SWATH-MS

① Principle: Based on data-independent acquisition, mass spectrometry data is collected by systematically scanning all predetermined mass windows.

② Advantages: Provides high-throughput, highly repeatable quantitative information, suitable for analysis of complex samples.

③ Disadvantages: Requires advanced data processing technology, high requirements for instrument performance.

 

(5) Multiple Reaction Monitoring (MRM)

① Principle: Specific precursor ion and product ion pairs are selected to quantify specific peptides or proteins.

② Advantages: Highly specific, high sensitivity, suitable for quantification of low-abundance target proteins.

③ Disadvantages: Can only quantify a limited number of target proteins at a time, requires prior knowledge of target protein information.