Streptavidin alkaline phosphatase vector sa 5100

One thousand cells were seeded on a glass coverslip in a 6 wells plate and incubated for 24 h at 37°C. Cells were washed twice with PBS and fixed with buffered formalin for 10 min, washed with PBS and treated with 0.5% Triton X-100 for 10 min at room temperature. After 30 min incubation with equine fetal serum as a blocking agent, cells were incubated for 2 h with an anti-BLV-p24 monoclonal antibody (1:500). Coverslips were washed three times with PBS and incubated with 0.5 µg/ml biotinylated anti-mouse IgG (Vector BA-9200) for 1 h. After three washes with PBS, coverslips were incubated with 1 µg/ml streptavidin-alkaline phosphatase (Vector SA-5100) for 30 min and washed with PBS. The reaction was developed by the addition of BCIP/NBT (Color Development Substrate, Moss Inc.) for 10 min, rinsed with water and stained with methyl green.

MAC-T, MAC-T BLV, and FLK cell culture supernatants were run in 12% polyacrylamide gels and transferred onto a Hybond + nylon membrane (Amersham Pharmacia, Sweden). After blocking with MTBS, the membrane was incubated 2 h with an anti-BLV-p24 monoclonal antibody (1:500) (a kind gift from Dr. Buehring, UCLA, Berkeley, USA) (Buehring et al., 1994) , washed 3 times for 15 min each with PBS-Tween and incubated with 0.5 µg/ml biotinylated anti-mouse IgG (Vector BA-9200) for 1 h. The membrane was washed with PBS-Tween and incubated with 1 µg/ml streptavidin-alkaline phosphatase (Vector SA-5100) for 30 min. After washing with PBS, BCIP/NBT (Color Development Substrate, Moss Inc.) was added for 10 min to develop the reaction and stopped by rinsing with tap water.