How to Detect Protein Expression in Western Blot Experiments
Detection of protein expression in Western Blot (WB) experiments:
Sample Preparation
1. Extract total proteins from the cells or tissues of interest.
2. Quantify the extracted proteins and prepare them in loading buffer suitable for SDS-PAGE analysis.
SDS-PAGE Gel Electrophoresis
1. Select an appropriate gel concentration based on the molecular weight of the target protein and cast the gel accordingly.
2. Load equal amounts of protein per lane and perform SDS-PAGE to separate proteins by molecular weight.
Membrane Transfer
1. Choose the appropriate transfer buffer and membrane (commonly polyvinylidene fluoride (PVDF) or nitrocellulose).
2. Transfer the separated proteins from the gel onto the membrane using a wet or semi-dry transfer method.
Blocking and Antibody Incubation
1. Block non-specific binding sites on the membrane using a suitable blocking buffer, such as 5% non-fat dry milk or bovine serum albumin (BSA).
2. Incubate the membrane first with a specific primary antibody against the target protein, followed by incubation with a secondary antibody that specifically recognizes the primary antibody.
Signal Detection
1. Depending on the conjugate of the secondary antibody, select an appropriate detection method (e.g., chemiluminescence or fluorescence).
2. Visualize the protein bands using an imaging system suitable for the selected detection method.
Data Analysis
1. Quantify the intensity of each protein band using image analysis software.
2. Conduct statistical analysis to assess the significance of differences in protein expression levels between experimental groups.
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