Can I make a stock solution of the crosslinker for long-term storage?
Stock solutions for long-term storage are NOT recommended because NHS esters are extremely vulnerable to hydrolysis. Even organic solvents are hygroscopic and tend to absorb water and promote hydrolysis. The crosslinker should be made immediately before each use.
How is the reaction quenched?
The reaction is quenched by the addition of 1 M Tris or glycine, pH 7.5 to the reaction mixture to a final concentration of 20-50 mM.
How much crosslinking reagent should I use?
We typically use a 10-fold molar excess of crosslinker to protein for solutions greater than 5 mg/ml. Use a 20- to 50-fold molar excess for more dilute protein solutions.
Is a hazy solution common after addition of the dissolved DSS solution to an aqueous buffer solution?
Yes. The resultant reaction mixture will become hazy frequently after the addition of the reagent solution.
Is this reagent water-soluble?
No. The reagent must first be reconstituted in an organic solvent before 10-fold dilution in aqueous buffers. The organic solvent should not exceed 10% of the total react volume.
What are the reactive groups and what are they reactive toward?
The reactive groups are NHS esters that are reactive toward primary amines.
What buffers are not recommended?
Any buffer containing primary amines (Tris, glycine, lysine, and ethanolamine) will not work! Glutathione- and imidazole-containing buffers are also not recommended.
What is the difference between DSS and BS3?
BS3 is the water-soluble analogue of DSS (i.e., DSS is the membrane-permeable analog of BS3).
What is the recommended buffer for optimal crosslinking?
We recommend the following buffer: 20 mM sodium phosphate, 0.15 M NaCl, PBS, pH 7.5. HEPES, bicarbonate-carbonate and borate buffers may be substituted for PBS. Nearly any non-amine buffer pH 7-8 can be used.
Will the crosslinked product become unlinked as a result of boiling, incubation with SDS, high salt, high or low pH or any other means?
No. DSS crosslinks are amide bonds (same as peptide bonds).