Team:TU Darmstadt/Protocols/Antibody staining

Antibody staining

Materials

• ice bath
• Centrifuge
• PBS buffer pH 7.4
• 1. antibody (mouse-anti-myc)
• 2. antibody (goat-antimouse-biotin)
• Streptavidin, R-phycoerythrin conjugate (SAPE)

Procedure

1. centrifuge 750 µL of cell suspension for 2 min in order to gain pellet
2. wash the pellet two times with 200 µL PBS buffer (after every wash centrifuge for 2 min)
3. add 30 µL of 1. antibody 1:10 concentration in PBS buffer
4. incubate suspension including antibody for 10 min. on ice
5. centrifuge for 2 min.
6. wash pellet three times with 200 µL PBS buffer (after every wash centrifuge for 2 min)
7. add 10 µL of 2. antibody 1:10 concentration in PBS buffer
8. incubate suspension including antibody for at least 30 min on ice
9. centrifuge for 2 min
10. wash pellet three times with 200 µL PBS buffer (after every wash centrifuge for 2 min)
11. add 10 µL of SAPE 1:10 concentration in PBS buffer
12. incubate on ice for 10 min.
13. wash two times with 200 µL PBS buffer

• now your cells are ready to be detected with FACS or fluorescent microscopy