Cell lysis, SDS-PAGE, and Western blotting were as previously described (Hames et al., 2005 (link)). For Western blotting, primary antibodies used were 1 µg/ml rabbit Nek6 (O’Regan and Fry, 2009 (link)), 1 µg/ml rabbit Nek7 (O’Regan and Fry, 2009 (link)), 0.3 µg/ml mouse α-tubulin (Sigma-Aldrich), 0.5 µg/ml mouse Flag (Sigma-Aldrich), 0.5 µg/ml mouse Hsp70 (Santa Cruz Biotechnology, Inc.), 0.5 µg/ml mouse Hsp72 (Enzo Life Sciences), 0.5 µg/ml mouse Hsc70 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit pHsp70 (this study), 0.8 µg/ml goat Nek9 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit GAPDH (Cell Signaling Technology), 1 µg/ml mouse phospho–histone H3 (EMD Millipore), 0.2 µg/ml rabbit γ-tubulin (Sigma-Aldrich), 0.4 µg/ml mouse TACC3 (Abcam), 0.5 µg/ml rabbit ch-TOG (Bethyl Laboratories, Inc.), and 1 µg/ml mouse Cep55 (Santa Cruz Biotechnology, Inc.). Nek7 antibodies were raised against a peptide corresponding to amino acid residues 12–28 conjugated to keyhole limpet hemocyanin via an N-terminal cysteine (C-OVPQFQPQKALRPDM). Secondary antibodies were HRP-labeled anti–mouse, anti–rabbit, or anti–goat IgGs (1:1,000; Sigma-Aldrich) or alkaline phosphatase–conjugated IgGs (1:7,500; Promega). HRP-labeled blots were detected by enhanced chemiluminescence (Thermo Fisher Scientific).
Rabbit γ tubulin
Manufactured by Merck Group
Rabbit γ-tubulin is a protein component of the cytoskeleton that plays a key role in the organization and nucleation of microtubules. It is a major structural constituent of the centrosome, which functions as the primary microtubule-organizing center in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Mouse α tubulin, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Mouse hsc70, by Santa Cruz Biotechnology (1 mentions)
Mouse hsp70, by Santa Cruz Biotechnology (1 mentions)
Mouse hsp72, by Enzo Life Sciences (1 mentions)
Mouse zo 1, by Thermo Fisher Scientific (1 mentions)
2 protocols using rabbit γ tubulin
1
Immunoblotting analysis of mitotic regulators
2
Immunofluorescence Staining of Dechorionated Embryos
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Dechorionated embryos were fixed overnight at 4 °C in PEM (80 mM Sodium-Pipes, 5 mM EGTA, 1 mM MgCl2) −4% PFA −0.2% Triton X-100. After washing 2 × 5 min in PEMT (PEM −0.2% TritonX100), 10 min in PEM −50 mM NH4Cl, 2 × 5 min in PEMT and blocking in PEMT −2% BSA, embryos were incubated 2 h at room temperature with primary antibodies. Following incubation, embryos were washed during 5, 10, 15, and 20 min in PEMT, blocked in PEMT −2% BSA, and incubated again with secondary antibodies for 2 h. Embryos were again washed during 5, 10, 15, and 20 min in PEMT, mounted in PEM −0.75% LMP-Agarose and analyzed on a Zeiss LSM710 laser scanning confocal microscope. The following primary antibodies were used in this study: Mouse@ZO-1 (1:250, Invitrogen 33-9100), Mouse@Acetylated Tubulin (1:2000, Sigma T7451), and Rabbit@γ-Tubulin (1:250, Sigma T5192). The secondary antibodies Goat@MouseIgG1-Alexa568, Goat@MouseIgG2b-Alexa647, and Goat@Rabbit-Alexa488 were obtained from Invitrogen and used at a dilution of 1:500.
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