β tubulin

Manufactured by Cell Signaling Technology

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β-tubulin is a structural protein that is a key component of microtubules, which are important cytoskeletal structures within cells. It plays a crucial role in cellular processes such as cell division, intracellular transport, and cell motility.

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Lab products found in correlation

Close spelling variants of this product

Anti-β-tubulin antibody (18 citations) Mouse anti-β-tubulin (16 citations) β-tubulin (9F3, (15 citations) β-tubulin antibody (11 citations) Rabbit anti-β-tubulin antibody (9 citations) Rabbit anti-α/β tubulin (8 citations) Anti-β-tubulin (2128S, (6 citations) Rabbit anti-β-Tubulin (#2128) antibody (3 citations) Mouse anti-β-tubulin antibody (3 citations) Anti-β-tubulin mouse monoclonal antibody (3 citations)

542 protocols using β tubulin

Western Blot Analysis of MC1R Protein

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For Western blot analysis of MC1R in total protein, adrenal or brain tissues were obtained. Total protein was extracted and electrophoresed. After transferring, the membrane was blocked in 5% bovine serum albumin and then incubated with the above MC1R antibody at 1:500. β-Tubulin was probed using anti–β-tubulin (Cell Signaling Technology #2146) as loading control.
For Western blot analysis of MC1R in membrane and cytosolic fractions, membrane and cytosolic fractions were obtained with a Membrane Protein Extraction Kit (Thermo Scientific, Waltham, MA; #89842). Na⁺/K⁺-adenosine triphosphatase (Cell Signaling Technology #3010) and β-tubulin (Cell Signaling Technology #2146) were probed as loading controls for membrane and cytosolic fractions, respectively.
To validate specificity of the MC1R primary antibody in vitro, B16 mouse melanoma cells were treated with short hairpin RNA (shRNA) construct targeting mouse MC1R (target sequence: 5′-AATGGAGATCAGGAAGGGATG-3′) according to published methods.^{28 (link)} Cells and reagents were kind gifts from Dr Rutao Cui, Boston University School of Medicine. Primary MC1R antibody was diluted at 1:1,000.

Chen X., Chen H., Cai W., Maguire M., Ya B., Zuo F., Logan R., Li H., Robinson K., Vanderburg C.R., Yu Y., Wang Y., Fisher D.E, & Schwarzschild M.A. (2017). The Melanoma-Linked “Redhead” MC1R Influences Dopaminergic Neuron Survival. Annals of neurology, 81(3), 395-406.

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Western Blot Analysis of IDH1 Mutant and Wild-Type

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Approximately 1x10⁷ cells (cell number determined by visual counting in hemocytometer) were lysed using cell lysis buffer (Cell Signaling, MA, USA) containing protease inhibitors (Millipore, MA, USA). Protein concentration was estimated using the Bradford method in order to load equivalent amounts of protein for western blotting. Proteins were resolved using polyacrylamide gel electrophoresis (BioRad) under denaturing conditions, followed by transfer to PVDF membranes (Millipore, MA, USA). Membranes were blocked overnight in 5% milk (Santa Cruz Biotechnology, TX, USA) in TBST (Tris-buffered Saline with 0.1% Tween 20, pH 7.5) at 4°C. Membranes were then washed 3 times for 5 min each in TBST and incubated with primary antibodies diluted in TBST [IDH1 wild-type (Cell Signaling, MA, USA), IDH1 R132H mutant (Dianova, Hamburg, Germany), β- tubulin (Cell Signaling, MA, USA)] for 1 h at room temperature. Following 3 washes of 10 min each with TBST, HRP-conjugated secondary antibodies (IDH1 mutant (Abcam, MA, USA), IDH1 wild-type and β- tubulin (Cell Signaling, MA, USA) were added for 1 h in TBST at room temperature. Membranes were washed thrice in TBST as described above and developed onto autoradiographic film using an enhanced chemiluminescence substrate kit (Thermo Scientific, MA, USA).

Izquierdo-Garcia J.L., Viswanath P., Eriksson P., Chaumeil M.M., Pieper R.O., Phillips J.J, & Ronen S.M. (2015). Metabolic Reprogramming in Mutant IDH1 Glioma Cells. PLoS ONE, 10(2), e0118781.

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Protein Concentration Measurement and Western Blot Analysis

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Protein concentration was measured by staining with amido black assay (Merck) according to the manufacturer's specifications. Per sample, 50 μg of proteins and 5 μl of PageRuler Prestained Protein Ladder (SM0671, Fermentas) were fractionated by electrophoresis through 12% SDS-polyacrylamide gels (SDS-PAGE) and transferred to Protran nitrocellulose membranes (Whatman) by semi-dry blotting using Tris-base, Glycerol, (Merck), 10 % SDS, methanol (J.T.Baker, The Netherlands) in double-distilled water for 30 min with a constant current at 100 mA per membrane. The membranes were blocked with 5% non-fat dry milk (AppliChem GmbH) in TBST (Tris-buffered saline (TBS) + 0.05% Tween^®-20, Serva) or with 3% BSA in TBST (Sigma-Aldrich, München). For the detection of active RAL and RAS proteins, we used the following primary mouse monoclonal antibodies: RALA included in STA-408-CB and pan-RAS included in STA-400-CB pull-down assays, RALB: #3523, KRAS: #2146 and beta-tubulin: #TA801672 (Cell Signaling). Anti-mouse IgG-Horseradish Peroxidase (HRP)-conjugate (1:5000 dilution, 325-035-045, Dianova) or anti-rabbit IgG-HRP-conjugate (1:10, 000 dilution; 7074, Cell Signaling) were used as secondary antibody. Protein bands were visualized using the ECL chemoluminescence detection system (GE Health Care) on X-ray films (Amersham Hyperfilm e).

Győrffy B., Stelniec-Klotz I., Sigler C., Kasack K., Redmer T., Qian Y, & Schäfer R. (2015). Effects of RAL signal transduction in KRAS- and BRAF-mutated cells and prognostic potential of the RAL signature in colorectal cancer. Oncotarget, 6(15), 13334-13346.

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Analyzing Extracellular Vesicle Proteins

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Transfected cells were washed in PBS and lysed directly into 4X Laemmli buffer (#1610747, Biorad, Watford, UK). Isolated EVs were lysed directly in 4X Laemmli buffer (#1610747, Biorad).Immunoblots were performed as previously described (46 (link)). All antibodies were purchased from Cell Signalling (NEB, Hitchin, UK) and were used at 1:1000 dilution: Dicer (D38E7, #5362S), Beta-Tubulin (9F3, #2128S), Annexin V (#8555S), Alix (3A9, #2171S), CD54/I-CAM (#4915S), EpCAM (D1B3, #2626S).

Probert C., Dottorini T., Speakman A., Hunt S., Nafee T., Fazeli A., Wood S., Brown J.E, & James V. (2018). Communication of prostate cancer cells with bone cells via extracellular vesicle RNA; a potential mechanism of metastasis. Oncogene, 38(10), 1751-1763.

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Subcellular Fractionation and Immunoblotting

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Whole cell lysates were collected in Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris, 1% Nonidet P-40). Isolation of cytosolic and nuclear fractions was done with an NE-PER kit (Pierce; Rockford, IL) following the manufacturer’s protocol. Protein (30 μg) was resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted overnight at 4 °C with primary antibodies. The various primary antibodies used were CTD110.6 (IgM, UAB), GLI1 (Cell Signaling; 2643), GLI2 (Boster Biological Technology; Pleasanton, CA; PA1941), Histone H3 (Cell Signaling; 4499), OGA (Santa Cruz Biotechnology; Dallas, TX; sc-376429), OGT (Sigma-Aldrich; O6264), PKM2 (Cell Signaling; 4053), phospho-Y105–PKM2 (Cell Signaling; 3827), and RL2 (Abcam; Cambridge, MA; ab2739). Alpha-tubulin (Cell Signaling; 12351) or beta-actin (Sigma-Aldrich; A3854) was used to confirm equal loading. Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (GE Healthcare; Chicago, IL) was used for detection, and blots were developed with either ECL or SuperSignal substrate (Pierce) and imaged on films or Amersham Imager 600. The purity of cytosolic and nuclear fractions was confirmed with beta-tubulin (2146; Cell Signaling) or histone deacetylase 1 (Cell Signaling; 2062) antibodies, respectively. The depicted images are representative of experiments repeated at least three times.

Das S., Bailey S.K., Metge B.J., Hanna A., Hinshaw D.C., Mota M., Forero-Torres A., Chatham J.C., Samant R.S, & Shevde L.A. (2018). O-GlcNAcylation of GLI Transcription Factors in Hyperglycemic Conditions Augments Hedgehog activity: Hyperglycemic conditions upregulate GLI activity. Laboratory investigation; a journal of technical methods and pathology, 99(2), 260-270.

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Subcellular Fractionation and Immunoblotting

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Western Blot Analysis of Protein Expression

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Tissue and cells were frozen in liquid nitrogen and homogenized in cell lysis buffer (20 mM HEPES-NaOH (pH 7.4), 25 mM KCl, 250 mM sucrose, 2 mM MgCl₂, 0.5 mM DTT and 1% digitonin (Sigma)) containing 1x Complete Protease Inhibitor and 1x PhosStop (Roche) using the Qiagen TissueLyser II. Tissue lysates were cleared by centrifugation, separated by SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk or 5% BSA in 1xTBS containing 0.1% Tween-20 for 1h at RT and incubated overnight with one of the following primary antibodies: rabbit anti-eGFP (GTX26556, 1:5000 in 5% milk in TBS-T), mouse anti-Lc (MA5-11860, 1:1000 in 5% milk in TBS-T), mouse-anti-GAPDH (sc-25778, 1:1000 in 5% milk in TBS-T), rabbit anti-CHC (Cell Signaling, 1:1000 in 5% BSA in TBS-T) or beta-Tubulin (Cell Signaling, 1:2000 in 5% BSA in TBS-T) followed by subsequent incubation with an HRP-conjugated secondary goat anti-rabbit or goat anti-mouse antibody (1:3000 in 5% milk or 5% BSA in TBS-T). Membranes were visualized using Clarity^™ Western ECL substrate (Biorad) and ChemiDoc^™ MP Imaging System (Biorad).

Grimm E., van der Hoeven F., Sardella D., Willig K.I., Engel U., Veits N., Engel R., Cavalcanti-Adam E.A., Bestvater F., Bordoni L., Jennemann R., Schönig K., Schiessl I.M, & Sandhoff R. (2022). A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis. PLoS ONE, 17(9), e0273660.

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Inflammation Signaling Pathway Inhibitors

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Resatorvid (TAK-242) and Ibudilast were purchased from MedChem Express (Monmouth Junction, NJ). ST-2825 was purchased from Apexbio Technology (Houston, TX). Carbenoxolone disodium was purchased from LKT Laboratories (St. Paul, MN). Most antibodies were purchased from Cell Signaling including phospho-NF-κB p65 (3033), phospho-p38 (9215), phospho-Akt (4060), phospho-JNK (9255), and beta tubulin (5666). The antibody for TLR4 western blots (sc-293072) was purchased from Santa Cruz Biotechnology (Dallas, TX), and the antibody for TLR4 IHC (ab22048) was purchased from Abcam (Cambridge, MA).

Janda J., Burkett N., Blohm-Mangone K., Huang V., Curiel-Lewandrowski C., Alberts D.S., Petricoin EF I.I.I., Calvert V.S., Einspahr J., Dong Z., Bode A.M., Wondrak G.T, & Dickinson S.E. (2016). Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin. Photochemistry and photobiology, 92(6), 816-825.

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Protein Extraction and Western Blot Analysis

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HOK cells were harvested and then lysed in a lysis buffer supplement with protease inhibitors (Beyotime Institute of Biotechnology, Haimen, China) and phosphatase inhibitors (Nanjing KeyGEN Biotech. Co., Ltd, China) for extraction of total cellular protein. The protein quantification was obtained by bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). For western blot analyses, 20-30 μg of total extracted proteins were injected into each lane before SDS-PAGE. Following transfer to polyvinylidene fluoride membranes, polyclonal anti-YOD1 (Proteintech, USA), anti-TGF-β3 (Proteintech), anti-Smad2/3 (Cell Signaling, Danvers, MA), and anti-phospho-Smad2/3 (Cell Signaling) were used for the detection of the protein expression levels. The expression of beta-tubulin (Cell Signaling) was used as an internal reference for protein loading.

Zhou X.L., Chen G., Li M.X., Wang H.X., Hong J.W., Shen J.Y., Wang Q., Ge X., Ding Z, & Xu L.C. (2018). Targeting YOD1 by RNA Interference Inhibits Proliferation and Migration of Human Oral Keratinocytes through Transforming Growth Factor-β3 Signaling Pathway. BioMed Research International, 2018, 6254308.

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Immunofluorescence and Western Blotting Antibodies

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Primary antibodies used for immunofluorescence microscopy and Western Blotting were obtained from Cell Signaling Technology and included: Beta-actin (#3700 and #4970), phospho-p70s6k T389 (#9234), NF kappa B p65 (#8242S), beta-tubulin (#2128S), Caspase-3 (#14220), Caspase-1 (#3866), Phospho-MLKL (#91689). Secondary antibodies used to detect the primary antibodies for Western Blotting were obtained from Thermo Fisher Scientific, and included goat anti-rabbit and goat anti-mouse IgG-HRP (#31460 and #31430). Secondary antibodies used for immunofluorescence microscopy were obtained from Molecular Probes (Life Technologies), and included goat anti-rabbit IgG AlexaFluor488 and goat anti-mouse IgG AlexaFluor594.

De-Leon-Lopez Y.S., Thompson M.E., Kean J.J, & Flaherty R.A. (2023). The PI3K-Akt pathway is a multifaceted regulator of the macrophage response to diverse group B Streptococcus isolates. Frontiers in Cellular and Infection Microbiology, 13, 1258275.

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