Alexa fluor 680 anti rabbit igg

Mouse anti-SAS6, rabbit anti-cortactin, and mouse anti-γ-tubulin: Santa Cruz; rabbit anti-STIL and mouse anti-TRIO: Abcam; rabbit anti-GEF-H1 and rabbit anti-integrin β1 (total form), mouse anti-GAPDH, rabbit anti-GOLPH2, rabbit anti-β-actin, rabbit anti-paxillin, mouse anti-γ-tubulin, and rabbit anti-NMIIA: GeneTex; mouse anti-α-tubulin and mouse anti-acetylated tubulin: Sigma-Aldrich; rat anti-α-tubulin: ABD; mouse anti-paxillin: BD; mouse anti-Rac1 and mouse anti-integrin β1 (active form): Millipore; rabbit anti-TRIO, Alexa Fluor 488 phalloidin, Alexa Fluor 488-anti-rat IgG, Alexa Fluor 488-anti-rabbit IgG, Alexa Fluor 488-anti-mouse IgG, Alexa Fluor 568-anti-mouse IgG, Alexa Fluor 568-anti-rabbit IgG, Alexa Fluor 680-anti-rabbit IgG, and DAPI: Thermo Fisher; and HRP-AffiniPure mouse anti-rabbit IgG and HRP-AffiniPure goat anti-mouse IgG: Jackson ImmunoResearch.

Cells were rinsed with cold PBS and lysed with hot 2 x Laemmli sample buffer. Proteins were separated by 12.5% SDS-PAGE, followed by electroblotting onto polyvinylidene fluoride (PVDF) membrane (Millipore, Etobicoke, ON). Membranes were blocked with 3% BSA/TrisHCl buffer saline (TBS)/pH 7.5 (blocking buffer), rinsed with 0.05% Tween 20/TBS (wash buffer) and probed primary antibodies/3% blocking buffer at room temperature overnight. The following day, membranes were washed 3 X 10 min, incubated 1 hour at room temperature with Alexa Fluor^® 660 anti-mouse IgG or Alexa Fluor^® 680 anti-rabbit IgG antibodies in wash buffer (ThermoFisher, Nepean, ON), washed and imaged using a LI-COR Odyssey Imager.