Thermo Scientific Pierce Crosslinking Technical Handbook
Thermo Scientific Pierce Crosslinking Technical Handbook
Thermo Scientific Pierce Crosslinking Technical Handbook
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Figure 3. Applications of Sulfo-SBED in protein interaction studies.<br />
Applications of Sulfo-SBED in Protein Interaction Studies<br />
B<br />
Protein 1 + sNHS – S-S ––––––– N 3<br />
Dark reaction<br />
pH 7.2<br />
B<br />
Protein 1 — S-S ––––––– N 3 + Protein 2<br />
UV 300-366 mm<br />
5-15 minutes<br />
B<br />
Protein 1 —– S-S ———— Protein 2<br />
Reduce<br />
Digest with Trypsin<br />
Protein 1 — SH + HS ——— Protein 2<br />
B<br />
SDS-PAGE<br />
B<br />
—– S-S ————<br />
Reduce<br />
B<br />
Protein 1<br />
Protein 2<br />
Western<br />
Transfer<br />
sA<br />
— SH + HS ——–<br />
Isolate biotin-containing<br />
fragment over immobilized<br />
streptavidin or monomeric<br />
avidin<br />
SH<br />
sA<br />
B<br />
Detect with: Streptavidin-HRP<br />
or Anti-Biotin Ab or Ab against<br />
Protein 2 with HRP-labeled<br />
secondary Ab.<br />
B<br />
Elute and separate<br />
via reversed-phase HPLC<br />
SH<br />
Western Blot<br />
Detection<br />
Sequence<br />
Analysis<br />
Mass Spec<br />
Identification<br />
Legend:<br />
sNHS<br />
S-S<br />
N3<br />
B<br />
Sulfo N-Hydroxy<br />
succinmide ester<br />
Disulfide bond<br />
Phenyl azide<br />
Biotin<br />
To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.<br />
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