Membrane-enriched whole-brain or hippocampal tissue homogenates (15–30 ug) were subjected to SDS-PAGE electrophoresis as previously described (Makinson et al., 2014 (link)). After blocking in 5% milk, blots were incubated overnight at 4°C in either polyclonal rabbit anti-Nav1.6 primary antibody (1:200, Millipore, Billerica, MA), polyclonal rabbit anti-Nav1.6 (1:225, Alomone, Israel), polyclonal rabbit anti-Nav1.1 (1:200, Millipore), or monoclonal mouse anti-Nav1.2 (1:1000, Neuromab, Davis, CA). Blots were then incubated in either HRP-conjugated goat anti-rabbit secondary (1:10,000, GE Healthcare, United Kingdom), HRP-conjugated goat anti-mouse secondary (1:10,000, Jackson ImmunoResearch, West Grove, PA), or HRP-conjugated goat anti-rabbit secondary (Sigma, St. Louis, MO, 1:16,000) for 1 hr followed by washing in SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher) and imaging. Blots were also probed using a monoclonal mouse anti-α-tubulin (1:10,000, Millipore) or monoclonal mouse anti-pan-cadherin (1:100,000, Sigma) antibody followed by HRP-conjugated goat anti-mouse secondary (1:10,000, Jackson ImmunoResearch) or HRP-conjugated goat anti-mouse secondary (Pierce, 1:26,000) for normalization of sample loading. Image quantification was performed using ImageJ software (NIH).
Mouse monoclonal anti α tubulin
Manufactured by Merck Group
Sourced in United States, Canada, United Kingdom, Germany
The Mouse monoclonal anti-α-tubulin is a laboratory reagent used for the detection and localization of α-tubulin, a key component of the cytoskeleton in eukaryotic cells. It is a mouse-derived monoclonal antibody that specifically binds to α-tubulin, allowing for its visualization and analysis in various cell biology and biochemical applications.
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Lab products found in correlation
Polyclonal rabbit anti nav1, by Merck Group (4 mentions)
Mouse monoclonal anti flag, by Merck Group (4 mentions)
Polyclonal rabbit anti nav1.6 primary antibody, by Merck Group (2 mentions)
Hrp conjugated goat anti mouse secondary, by Jackson ImmunoResearch (2 mentions)
Hrp conjugated goat anti rabbit secondary, by Merck Group (2 mentions)
Monoclonal mouse anti pan cadherin, by Merck Group (2 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (2 mentions)
Mouse monoclonal anti gfp, by Roche (2 mentions)
Odyssey near infrared scanner, by LI COR (2 mentions)
Enhanced chemiluminescence, by GE Healthcare (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Mouse monoclonal anti smn, by BD (2 mentions)
Peroxidase affinipure goat anti mouse igg h l wb, by Jackson ImmunoResearch (2 mentions)
Mouse monoclonal anti β catenin, by BD (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Mouse monoclonal anti acetylated α tubulin, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Phosstop, by Roche (2 mentions)
Anti γh2ax s139 20e3, by Cell Signaling Technology (2 mentions)
79 protocols using mouse monoclonal anti α tubulin
1
Quantitative Immunoblotting of Ion Channels
2
Western Blot Analysis of Sodium Channels
3
Western Blot Analysis of Protein Samples
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Tissue sample was ground in liquid nitrogen and total proteins were extracted using 2 × SDS loading buffer. The samples were resolved on a 12% SDS–PAGE gel and transferred onto nitrocellulose membranes using a Tris-Glycine transfer buffer. The blots were probed with the appropriate antibodies: monoclonal mouse anti-GFP, which also recognizes YFP (Roche, 11814460001, 1:2,000 dilution); monoclonal mouse anti-FLAG (Sigma-Aldrich, F3165, 1:2,000 dilution); monoclonal mouse anti-α tubulin (Sigma-Aldrich, T6074, 1:4,000 dilution); polyclonal rabbit anti-PR1 (obtained from Xinnian Dong68 (link), 1:2,000 dilution); polyclonal rabbit anti-SE (serum containing polyclonal antibodies was produced in rabbits immunized with peptide containing the first 200 amino acids of the SE protein, AbMax Biotechnology Co., Ltd., 1:1,000 dilution); rabbit serum was purified using Montage Antibody Purification kit, Millipore); goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, sc-2005, 1:4,000 dilution); and goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, sc-2030. 1:4,000 dilution).
4
Mycosubtilin Activates MAPK Signaling
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Seedlings grown on Murashige and Skoog medium for two weeks were sprayed with μ10 g/mL of mycosubtilin homologue. Samples were collected at 0, 5, 10, and 30 min and were analyzed by Western blotting using monoclonal rabbit phospho-p44/42 MAPK (Erk1/2) XP antibodies (Cell Signaling Technology, Danvers, MA, USA) (1:2,000 dilution). Detected with a monoclonal mouse anti-α-tubulin (Sigma-Aldrich) (1:4,000 dilution).
5
Protein Extraction and Western Blot Analysis
6
Flg22-Induced MAPK Activation Dynamics
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Two-week-old seedlings grown on half-strength Murashige and Skoog medium were treated with 10 μM flg22 containing 0.01% Silwet L-77. Samples were collected at 0, 5, 10, 15 and 30 min, and analysed by western blotting using monoclonal rabbit phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)(D13.14.4E) XP antibodies (Cell Signaling Technology, #4370S, 1:2,000 dilution). α-tubulin was used as a loading control using monoclonal mouse anti-α tubulin (Sigma-Aldrich, T6074, 1:4,000 dilution).
7
Western Blot Analysis of Aortic Proteins
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Solubilized proteins from aortic sarcolemma were separated by 10% Tris‐glycine polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare/Life Sciences). The proteins were visualized using polyclonal rabbit anti‐PKG1 (cGPK1β) antibody (Stressgen, Victoria, BC, Canada, 1:2000), rabbit polyclonal anti‐Fli1 antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), goat polyclonal anti‐collagen‐1 antibody (1:500; Southern Biotechnology, Birmingham, AL) followed by incubation with peroxidase‐conjugated anti‐mouse, anti‐rabbit antiserum (Amersham Corp., 1:1000), or donkey anti‐goat antiserum (Santa Cruz Biotechnology, Inc., 1:1000). Bands were visualized by 1‐ to 30‐minute exposure of nitrocellulose membrane to Premium Blue X‐ray film (Phenix Research Products at Thomas Scientific, Swedesboro, NJ), and optical density was estimated by laser densitometry (Kodak Molecular Imaging Software, version 5.0). To normalize levels of proteins against levels of GAPDH or α‐tubulin, membranes were stripped and reprobed with rabbit monoclonal anti‐GAPDH antibody (Cell Signaling Technology, Danvers, MA; 1:1000) or with monoclonal mouse anti‐α‐tubulin (Sigma‐Aldrich, St. Louis, MO; 1:5000).
8
Immunostaining of 2D cell cultures
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2D cultures stainings were fixed using 4% paraformaldehyde or methanol/acetone (1:1) depending on the antibody. Cells were permeabilized with 0.1% Triton X-100 in PBS and incubated in blocking solution (5% horse serum or 0.2% gold fish gelantine). Cells were subsequently incubated with 1:1000 monoclonal mouse anti-acetylated-Tubulin antibody (Sigma-Aldrich, Saint Louis, USA), 1:200 monoclonal mouse anti-α-Tubulin (Sigma-Aldrich, Saint Louis, USA), 1:200 mouse anti-γ-Tubulin (Sigma-Aldrich, Saint Louis, USA), 1:200 rabbit-anti-Ift88 (ProteinTech Group Inc, Chicago, USA), 1:100 goat anti-Scrib (C20, SantaCruz, Dallas, USA), 1:200 monoclonal mouse anti-Golgin-97 (Molecular Probes Europe, Leiden, The Netherlands), 1:200 rabbit anti-GM130 (Sigma-Aldrich, Saint Louis, USA), anti rabbit-Cep164 (gift from Erich A. Nigg) and Hoechst 33342 (Life Technologies GmbH, Darmstadt, Germany). Antibodies were visualized using Cy3-, Cy5- or Alexa-488-labelled secondary antibodies at a dilution of 1:1000 (Jackson Immunoresearch, West Grove, USA).
9
Quantifying Luciferase Expression in Transfected Promastigotes
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Whole cell lysates were prepared by lysing 1x107 promastigotes from each transfectant in 100 μl of 1x Laemmli’s buffer [56 (link)]. 10 μl lysate was used to fractionate proteins on 12% SDS-PAGE. Fractionated proteins were electroblotted on nitrocellulose membranes (Whatman) and probed sequentially with polyclonal goat anti-luciferase (Promega) and monoclonal mouse anti-α-tubulin (Sigma). The labeling was visualized with horseradish peroxidase-conjugated mouse anti-goat (Pierce) and rabbit anti-mouse (Abcam) respectively using a Western Lightning Chemiluminescence Reagent Plus system (PerkinElmer). Amount of luciferase expression relative to cells transfected with pSPYNEOαLUC was estimated by densitometric analysis using ImageJ software followed by normalization against the amount of α-tubulin of the respective cells. Error bars were calculated from the mean ± SE of two independent experiments.
10
Western Blot Analysis of ICAM-1 and CD36
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Cells were lysed in boiled Sample Buffer 1x (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 100 mM DTT). In total, 30 μg of total protein extracts were resolved on 8% SDS-PAGE and electrically transferred onto poly(vinylidene difluoride) membranes (PVDF, Bio-Rad Laboratories, Hercules, CA, USA) membranes. Membranes were blocked with TBS-T (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.02% Tween-20) containing 5% skimmed milk (Bio-Rad), for 1 h at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in TBS-T containing 5% milk. The following antibodies were used: mouse monoclonal anti-ICAM-1 (Santa Cruz Biotechnology, Dallas, TX, USA; working dilution 1:500), rabbit polyclonal anti-CD36 (Santa Cruz Biotechnology, working dilution 1:500), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, working dilution 1:10,000). After extensive washing, immunecomplexes were detected with horse-radish peroxidase conjugated species-specific secondary antibodies (Jackson Laboratory, Bar Harbor, ME, USA) followed by enhanced chemiluminescence reaction (Millipore Corporation, Billerica, MA, USA). Proteins detected by immunoblotting were quantified by densitometry (ChemiDoc imaging system, BioRad) and normalized as a function of α-tubulin with Image-Lab 5.0 software (Bio-Rad).
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