Cells were allowed to settle on poly-L-lysine coated coverslips (BD Biosciences) at 37°C, fixed with 4% paraformaldehyde (USB Corporation) or methanol, permeabilized with 1X Dako wash buffer (Dako) and blocked with 20% normal goat serum (Invitrogen) or donkey serum (Abcam) in 1X Dako wash buffer. Primary antibody incubation was overnight at 4°C. The following primary antibodies were used: rabbit anti-Numb 1:50 or 1:100 (Abcam), goat anti-LIS1 1:500 (Santa Cruz Biotechnology), mouse anti-alpha-tubulin 1:200 (Abcam), rat anti-α-tubulin 1:1000 (Abcam), mouse anti-α-tubulin conjugate FITC 1:200 (Sigma). Secondary antibody incubation was performed for 1 hr at room temperature. DAPI (Molecular Probes) was used to detect DNA. Images were obtained with a Confocal Leica TCS SP5 II (Leica Microsystems) or an Axio Observer.Z1 microscope with the LSM 700 scanning module (Zeiss). ImageJ 1.46r was used to determine fluorescence intensity.
Rat anti α tubulin
Manufactured by Abcam
Sourced in United States
Rat anti-α-tubulin is a primary antibody that specifically binds to the α-tubulin protein, a component of the cytoskeleton. This antibody can be used in various applications to detect and analyze α-tubulin expression and distribution within cells.
Automatically generated - may contain errors
Lab products found in correlation
Normal goat serum (ngs), by Thermo Fisher Scientific (2 mentions)
Poly l lysine coated coverslips, by BD (2 mentions)
Dako wash buffer, by Agilent Technologies (2 mentions)
Rabbit anti numb, by Abcam (2 mentions)
Goat anti lis1, by Santa Cruz Biotechnology (2 mentions)
Mouse anti alpha tubulin, by Abcam (2 mentions)
Mouse anti α tubulin conjugate fitc, by Merck Group (2 mentions)
Tcs sp5 2 confocal, by Leica (2 mentions)
Axio observer z1 microscope, by Leica (2 mentions)
Lsm 700 scanning module, by Zeiss (2 mentions)
Donkey serum, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Hoechst, by Thermo Fisher Scientific (2 mentions)
Prime western blotting detection reagent, by GE Healthcare (1 mentions)
Rabbit anti gfp, by Cell Signaling Technology (1 mentions)
Rabbit anti tdp43, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail without edta, by Roche (1 mentions)
Mouse anti α actinin, by Merck Group (1 mentions)
6 protocols using rat anti α tubulin
1
Immunocytochemistry of Cytoskeletal Proteins
2
Quantitative Western Blot Analysis
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A 15 μg aliquot of protein per sample was resolved on a denaturing 10% polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a semidry blotter by established standard protocols (Figueroa-Romero et al., 2009 (link); Lunn et al., 2009 (link)). Blots were probed overnight at 4°C with the following primary antibodies: rabbit anti-TDP43 (1:1,000; cat#A260, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-histone 2B (H2B) member S (1:500; cat# NB100-56347, Novus Biologicals, Littleton, CO, USA), rabbit anti-GFP (1:1,000; cat#2555, Cell Signaling Technology), and rat anti-α-tubulin (1:5,000; cat#ab6160, Abcam). The next day, blots were rinsed and probed for 50 min at room temperature with appropriate HRP-conjugated secondary antibodies (1:1,000; Santa Cruz Biotechnology or New England Biolabs), then rinsed and visualized by enhanced chemiluminescence with Prime Western Blotting Detection Reagent (Amersham, GE Healthcare, Chicago, IL, USA).
3
Immunocytochemistry of Cytoskeletal Proteins
4
Western Blot Analysis of Knockdown Cells
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For analysis of knockdown cultures cells were incubated in 400 µL lysis buffer (50 mM HEPES pH 7.5, 0.5% NP-40, 5% Triton X-100, 50 mM NaCl, 5% Glycerol, 1 mM EDTA, and protease inhibitor cocktail without EDTA [Roche]) on ice for 10 min. Lysates were sonicated for 30 sec and cleared by centrifugation at 13,000 rpm. An amount of 30 µg total protein was separated on 4%–12% NuPAGE gels and transferred on poly-vinylidene difluoride (PVDF) membrane with 1× transfer buffer (25 mM Tris Base and 192 mM Glycine) supplemented with 5% methanol. Antibody dilution for primary rabbit anti-hPcf11 (Abcam) was 1:2000, rat anti-α-tubulin (Abcam ab 6160) was 1:2000, rabbit anti-CPSF73 (Abcam) was 1:2000; secondary HRP coupled anti-rabbit antibody (Abcam) was used 1:5000 and HRP coupled anti-rat antibody (Abcam) was used 1:5000.
5
Multicellular Immunofluorescence Staining Protocol
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All cell types were fixed with 4% paraformaldehyde (45 min) and treated with blocking buffer for 45 min. Only fixed nRCMs were treated with permeabilisation buffer (3% Triton X-100) for 10 minutes preceding blocking procedure. After blocking, nRCMs were incubated for 3 hours at 37 °C with the primary antibodies mouse anti-α-Actinin (1:1000, Sigma), and rat anti-α-Tubulin (1:1500, Serotec). NRCFs were treated with rabbit anti-Collagen 1α1 (1:500, Abcam) and rat anti-α-Tubulin (1:750) antibody overnight at 4 °C. BMDMs were treated with rabbit NFκB p65 antibody (1:300 Santa Cruz #sc-372) and rat anti-α-Tubulin (1:750) overnight at 4 °C. After primary antibody incubation, nRCMs and nRCFs were incubated with secondary antibodies goat anti-rat AlexaFluor555 (1:750, Life Technologies), goat anti-mouse AlexaFluor488 (1:500, Life Technologies) and Hoechst (1:6000, Invitrogen) for 1 hour at room temperature. BMDMs were incubated with secondary antibodies goat anti-rat AlexaFluor555 (1:500), goat anti-rabbit AlexaFluor488 (1:750, Life Technologies) and Hoechst for 1 hour at room temperature.
6
GEF-H1 Regulation in Cytoskeleton Dynamics
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fMLP, blebbistatin, anti–human IgG F(ab')2, and recombinant human TNF were obtained from Sigma-Aldrich. C5a and ICAM-1/Fc chimera were from R&D Systems. Nocodazole and Y-27632 were obtained from EMD Millipore. Latrunculin A was obtained from Alexis Corporation.
Antibodies used for immunoblotting were mouse anti-MLC (1:2,000; Sigma-Aldrich), rabbit anti-pMLC (1:500; Cell Signaling Technology), sheep anti–GEF-H1 (1:500; Exalpha Biologicals), and mouse anti-GAPDH (1:26,000; Sigma-Aldrich).
Antibodies used for immunofluorescent staining were mouse anti–GEF-H1 (1:50; Hycult), rabbit anti–GEF-H1-pS885 (1:200; Cell Signaling Technology), rabbit anti–flotillin-2 (1:100; Santa Cruz Biotechnology, Inc.), rat anti–α-tubulin (1:100; Abcam), and mouse anti-pMLC (1:50; Cell Signaling Technology).
Antibodies used for immunoblotting were mouse anti-MLC (1:2,000; Sigma-Aldrich), rabbit anti-pMLC (1:500; Cell Signaling Technology), sheep anti–GEF-H1 (1:500; Exalpha Biologicals), and mouse anti-GAPDH (1:26,000; Sigma-Aldrich).
Antibodies used for immunofluorescent staining were mouse anti–GEF-H1 (1:50; Hycult), rabbit anti–GEF-H1-pS885 (1:200; Cell Signaling Technology), rabbit anti–flotillin-2 (1:100; Santa Cruz Biotechnology, Inc.), rat anti–α-tubulin (1:100; Abcam), and mouse anti-pMLC (1:50; Cell Signaling Technology).
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