I have put together a protocol below to crosslink the proteins that is potentially bound with my nuclear protein of interest. Please reply with suggestions and your experiences.
1. Dissolve DSP in dry DMSO at a 20mM concentration.
2. Dilute the DSP solution 1:10 in dry DMSO (1ml of 20mM DSP with 9ml solvent (PBS?) to make 2mM DSP.
3. Wash cells (in culture plates or harvested in micro centrifuge tubes?) twice with Phosphate Buffered Saline (PBS; e.g., 0.1M phosphate, 0.15M NaCl; pH 7.2) to remove media.
5. Add the Crosslinker Solution to a final concentration of 2mM.
6. Incubate the reaction mixture at room temperature for 30 minutes or on ice for 2 hours.
7. Add the Stop Solution (1M Tris, pH 7.5) to a final concentration of (10-20mM?) and incubate for 15 minutes.
After Immunoprecipitation – to analyze bound proteins on western blot cleave DSP bonds
To cleave DSP use (20-50mM DTT at 37°C for 30 minutes or 2-mercaptoethanol in 2% SDS, 62.5mM Tris base, 10% glycerol at 100°C for 5-15? minutes?)
If you cleave DSP, the lysine residues open up and the antibodies should detect the proteins?
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