Tubulin gamma (mouse)

Tubulin gamma (mouse) kdorfman Tue, 10/02/2018 - 17:17

binds to centrosomes

From Pat
Monoclonal, Sigma clone GTU-88
Fix in paraformaldehyde or cold methanol
Dilute 1:100

From Drew Sigma T6557-100UL

  • Working concentration is 1:5000
  • Frozen stock is diluted 1:1 with glycerol
  • 5 uL aliquots (in half-mL tubes) should be diluted:
    • 1:50 in the tube: add 245 uL PBS-Tw-BSA to make 1:100 stock
    • 1:50 for use (1 uL 1:100 antibody + 49 uL PBS-Tw-Az-BSA per coverslip)

Drew's Protocol:

  • Rinse in PBS
  • Fix in paraformaldehyde 15 minutes
  • Rinse with PBS-Triton
  • Rinse with PBS-Tween
  • Rinse with PBS-BSA 1 hour
  • Dilute Ab 1:5000 in BSA
  • Incubate with Ab 2hr RT
  • Rinse with PBS 3x
  • Incubate with secondary Ab (in BSA)
  • Rinse with PBS 3x
  • Mount

Test Protocol:

  • coverslips already fixed in paraglut, stored in PBS-Tw-Az 1
  • 1 uL Ab (as supplied by Sigma) into 99 uL PBS-BSA (save!)
  • 1 uL 1:100 Ab into 499 uL PBS-BSA (save for the rest of the slides if it works!)
  • 50 uL onto coverslip - 2 hr RT incubation
  • rinse with PBS-Tw-Az
  • 50 uL Goat anti mouse 488

  1. Results: some background fluorescence. Add a blocking step:
    rinse 30 min in 2% BSA
    rinse 2x in PBS-Tw-Az
    Didn't make much difference. centrosomes are small and bright ↩︎