How to Detect Target Proteins by Western Blot
Western blotting quantitatively analyzes the expression levels of target proteins by separating protein samples through SDS-PAGE, transferring them to membranes, and subsequently binding and detecting with specific antibodies. The general procedure includes sample preparation, protein quantification, electrophoretic separation, membrane transfer, antibody binding, and detection. Detailed steps and important considerations are as follows:
1. Sample Preparation
Collect cell or tissue samples and lyse them to release proteins, using lysis buffers such as cell lysis buffer or RIPA buffer. Add protease and phosphatase inhibitors to the lysis buffer to prevent protein degradation and dephosphorylation. Apply sonication to disrupt cells or tissues thoroughly, followed by centrifugation to obtain a clear lysate.
2. Protein Quantification
Quantify protein concentrations in the lysate using assays such as the BCA assay, Lowry assay, or Bradford assay. Dilute protein samples to uniform concentrations as necessary.
3. SDS-PAGE Electrophoresis
Mix protein samples with sample buffer containing reducing agents and SDS. Heat the samples to denature proteins and disrupt secondary structures. Load the samples into wells of a polyacrylamide gel (SDS-PAGE) and perform electrophoresis to separate proteins based on molecular weight. Adjust gel concentrations and electrophoresis conditions as needed to achieve optimal separation of proteins of different sizes.
4. Protein Transfer
Transfer separated proteins from the gel onto a polyvinylidene fluoride (PVDF) membrane or a nitrocellulose (NC) membrane. Use either wet or semi-dry transfer methods, ensuring proper conditions (such as electric current and transfer time) for complete protein transfer.
5. Blocking and Antibody Binding
Block unoccupied binding sites on the membrane using non-specific proteins, such as bovine serum albumin (BSA), to minimize non-specific binding. Incubate the membrane in antibody buffer containing target protein-specific primary antibodies to allow specific binding. Optimize incubation time and temperature according to the properties of the antibody and target protein.
6. Secondary Antibody Binding and Detection
Incubate the membrane with a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP), targeting the species of the primary antibody (e.g., anti-rabbit IgG or anti-mouse IgG). Allow the secondary antibody to bind specifically to the primary antibody. Detect bound antibodies using chemiluminescent or chromogenic methods to generate visualized signals.
7. Data Analysis
Quantify Western blot signals using image analysis software such as ImageJ or Quantity One. Determine the expression levels of the target protein by comparing band intensities across different samples. Use internal reference proteins, such as β-actin or GAPDH, as loading controls to normalize sample variations.
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