Cells were washed with ice-cold PBS three times and treated with 1–5mM disuccinimidyl suberate (DSS, A39267, Thermo Scientific, Waltham, MA) for 30 min at room temperature. The reaction was stopped by adding the quenching solution (1M Tris, PH 7.5) to the final concentration of 20mM for 15 min. Then, cell lysates were used for WB as described above.
Protocol detail
Crosslinking Cells with DSS
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Corresponding Organization : Louisiana State University Health Sciences Center Shreveport
Other organizations : Overton Brooks VA Medical Center
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Variable analysis
independent variables
- Concentration of disuccinimidyl suberate (DSS) (1–5mM)
- Protein levels/interactions as measured by Western blot (WB)
- Washing with ice-cold PBS three times
- Reaction time with DSS (30 min)
- Reaction temperature (room temperature)
- Quenching time (15 min)
- Quenching solution (1M Tris, pH 7.5)
- Positive control: Not specified
- Negative control: Not specified
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