Labeling of streptavidin
Streptavidin (sa) is now more commonly used as an enzyme conjugate labeled with streptavidin because of its low positive surface charge and the absence of glycans, which makes it much less non-specific to bind than avidin.
.Preparation of HRP-SA conjugate Direct labeling with sodium periodate: the reaction was carried out at 4, Naio4 was added to HRP first, then directly mixed with streptavidin solution, and then mixed with carbonate buffer (pH 90) After dialysis, the reaction was terminated with KBH4: saturated ammonium sulfate precipitation was added, the supernatant was discarded after centrifugation, and the HRP-SA conjugate was obtained by reconstitution and precipitation with PBS
.Preparation of SA biotinylated HRP complex Biotinylated HRP (HRP-B) was prepared by BNHS-labeled antibody method, and then HRP-B was appropriately diluted, and an equal volume of streptavidin solution (HRP-B and SA concentration ratio of about 1:4) was added to react to obtain SA biotinylated enzyme complex (STREPT**idin-biotin-peroxidaes complex, SABC).
.Preparation of AP-SA conjugate Glutaraldehyde two-step method: first, use saturated ammonium sulfate to precipitate alkaline phosphatase, add excess glutaraldehyde solution to reconstitute alkaline phosphatase after centrifugation One aldehyde group of glutaraldehyde is combined with the amino group of alkaline phosphoryl alcohol: after full dialysis to remove unbound glutaraldehyde, add streptavidin to bind another aldehyde group of glutaraldehyde bound to alkaline phosphatase, terminate the reaction with lysine, and leave the supernatant containing AP-SA conjugate after centrifugation for later use.