Team:TU Darmstadt/Protocols/Antibody staining

From 2012.igem.org

(Difference between revisions)
(Materials)
(Procedure)
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# incubate on ice for 10 min.
# incubate on ice for 10 min.
# wash two times with 200 µL PBS buffer  
# wash two times with 200 µL PBS buffer  
-
* now your cells are ready to be detected with FACS or fluorescent microscopy
+
* * now your cells are ready to be detected via flow cytometry

Revision as of 16:59, 23 September 2012

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 via flow cytometry