Principles and Steps of Western Blotting, Differences with ELISA, and Choosing the Right Method for Protein Quantification
Western Blotting
1. Experimental Principle
Western Blotting is a technique used to detect the presence and expression levels of specific proteins in samples. The basic principle involves separating proteins using SDS-PAGE electrophoresis, transferring the proteins to a membrane (e.g., PVDF), and then detecting the target protein using specific antibodies.
2. Experimental Steps
(1) Protein Sample Preparation: Extract proteins from cells or tissues.
(2) SDS-PAGE Electrophoresis: Use electrophoresis to separate proteins in the sample.
(3) Transfer: Transfer the electrophoretically separated proteins to a membrane.
(4) Blocking: Use non-specific proteins, such as BSA, to block unbound sites on the membrane to prevent non-specific binding.
(5) Incubation: First, use a primary antibody to identify the target protein, then incubate with a secondary antibody (which binds to the primary antibody and is often labeled with fluorescence or enzymes).
(6) Detection: Use a fluorescence detector or developer solution to detect the signal of the target protein.
ELISA (Enzyme-Linked Immunosorbent Assay)
1. Experimental Principle
ELISA is an enzyme immunoassay technique used to detect antigen or antibody concentrations. The core principle is based on the specific antibody-antigen reaction, combined with enzyme activity to amplify the detection signal.
Differences Between Western Blotting and ELISA
1. Purpose
Western Blotting is mainly used to detect the presence and expression levels of specific proteins, while ELISA is primarily used to measure the concentration of specific antigens or antibodies.
2. Sample Type
Western Blotting detects separated proteins, while ELISA can directly measure antigens or antibodies in unprocessed samples.
3. Sensitivity
ELISA is generally more sensitive and can detect very low concentrations of antigens or antibodies.
4. Output
Western Blotting produces a protein band pattern, while the output of ELISA is usually a light absorbance value.
Selection Recommendations
1. Protein Expression Verification
Recommended to use Western Blotting.
2. Quantitative Measurement
For antibody concentration determination in serum, ELISA is recommended.
3. Rapid Screening of Multiple Samples
ELISA is recommended.
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