Method Using a DSS Crosslinker for IP and Polymeric Protein WB
DSS (Disuccinimidyl Suberate) crosslinker is commonly used to stabilize protein-protein interactions, thereby enhancing the efficiency of immunoprecipitation (IP) and subsequent Western Blot (WB) analysis. Below is a protocol for performing IP and crosslinked protein WB analysis using DSS:
Crosslinking
1. Sample Preparation
Extract proteins from cells or tissues and adjust protein concentration.
2. DSS Addition
Add DSS crosslinker to the protein sample. DSS is a widely used reversible crosslinker that forms covalent bonds between lysine residues in proteins.
3. Incubation
Incubate at room temperature or the appropriate temperature for a defined period, typically between 30 minutes to several hours, depending on the experimental requirements and the desired crosslinking efficiency.
4. Reaction Termination
Terminate the crosslinking reaction by adding Tris buffer (usually 1M, pH 7.5).
Immunoprecipitation (IP)
1. Lysate Preparation
Use an appropriate lysis buffer to prepare cell lysates and remove insoluble components.
2. Pre-Clearing
Pre-clear the lysate with A/G beads to eliminate nonspecific bindings.
3. Antibody Incubation
Add a specific antibody targeting the protein of interest and incubate overnight at 4°C.
4. Binding to Protein A/G Beads
Incubate the antibody-bound lysate with Protein A or G beads to capture the antibody-antigen complex.
5. Washing and Pelleting
Wash the beads several times with IP buffer to remove unbound proteins and other impurities.
6. Elution
Elute the captured antigen-protein complex using SDS sample buffer.
Western Blot (WB)
1. Sample Preparation
Heat the eluted proteins from the IP procedure and perform electrophoresis on an SDS-PAGE gel.
2. Protein Transfer
Transfer the separated proteins from the gel onto a PVDF or nitrocellulose membrane.
3. Blocking
Block the membrane with 5% skim milk or BSA to prevent nonspecific binding.
4. Antibody Detection
Incubate with the primary antibody targeting the protein of interest, followed by incubation with an HRP-conjugated secondary antibody.
5. Signal Detection
Detect the signal using chemiluminescent substrate and capture the signal using X-ray film or a digital imaging system.
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