Tubulin gamma (mouse)

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