γ Tubulin Antibody (TU-30): sc-51715

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:500) -Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

Cells were transfected for 48 h, prior to the addition of cell lysis buffer to extract proteins. The homogenate was then centrifuged at 700 × g for 5 min to pellet large cellular debris. The samples were heated at 100°C for 5 min then loaded onto 15% SDS-PAGE for electrophoresis. For the western blot analysis, the separated proteins were transferred to Protran nitrocellulose (Micron separations, Inc, Westborough, MA, USA). The blots were blocked in blocking buffer [1% phosphate-buffered saline (PBS), 0.1% Tween 20 and 3% bovine serum albumin) and then incubated with rabbit anti-STK17A antibody (SRP00679; Saier Corporation, Tianjin, China) at a 1:100 dilution, γ-tubulin acted as an internal standard that detected by mouse anti γ-tubulin (sc-51715; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 1:500 dilution. The primary antibodies were detected with secondary goat anti-rabbit IgG (diluted 1:2,000 in PBS containing Tween 20; A0545; Sigma-Aldrich) conjugated with horseradish peroxidase (Sigma-Aldrich). Labeled bands from the blots were detected by enhanced chemiluminescence (Beyotime Institute of Biotechnology, Haimen, China). The bands intensities were quantified by LabWorks software 4.0 as previously described.

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