α tubulin

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α-tubulin is a protein that is a major structural component of microtubules, which are cytoskeletal filaments found in eukaryotic cells. α-tubulin plays a crucial role in the formation and function of the mitotic spindle, which is essential for cell division.

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433 protocols using α tubulin

UVB-Induced Transcriptional Regulation in MCF7 Cells

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MCF7 cells were plated in 6 cm plates prior treatment. At 80–90% confluence, cells were treated with 55 J/m² UVB. The irradiated and control cells 1, 2, 3, 4, 5, 6, hours later after incubation were washed twice with ice-cold PBS containing complete protease inhibitor cocktail (1×), phenylmethylsulfonyl fluoride (PMFS)(0.5 mM), β-glycerophosphate (10 mM), sodium orthovanadate (1 mM), sodium fluoride (20 mM) inhibitors. Cells were scraped in PBS containing protease inhibitor cocktail and frozen-thawed three times. Samples were sonicated for 10 cycles (30 second on, and 30 seconds off) (Bioruptor) then boiled for 5 min in loading solution. Protein samples were separated by a 6–10% SDS-PAGE, transferred and western blot assays were carried out by using standard methods. The following antibodies were used: RNA polymerase II N-terminal H-224× (Santa Cruz), Pol II Ser2, AB: Covance, (MMS-129R), Pol II Ser5 AB: Abcam, (ab5131) Pol II Ser 7 AB: [64] (link), TBP: 3G3 [65] (link), TFIIH subunit (p62): 3C9MAB [66] (link), TFIIB: [67] (link), CDK7: (C-19), sc-529 (Santa Cruz), Tubulin-α: (D-10): sc-135659 (Santa Cruz), GAPD: 6C5 (MAB374, Millipore), CSB: 1CSB- 1A11 [68] (link). In Figure S1 Phospho-p53 (Ser15) antibody #9284 (Cell Signaling Technology); Phospho-Chk1 (Ser345) (133D3) antibody #2348 (Cell Signaling Technology); Tubulin-α: (D-10) antibody: sc-135659 (Santa Cruz) were used.

Gyenis Á., Umlauf D., Újfaludi Z., Boros I., Ye T, & Tora L. (2014). UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells. PLoS Genetics, 10(7), e1004483.

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Polyclonal Antibody Production and Validation

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The polyclonal antibody to the Vav1 DH domain (Ref. 302-5) was raised in rabbits using a maltose binding protein-Vav1 DH fusion protein previously purified from Escherichia coli according to standard techniques. Polyclonal antibodies to the indicated Vav1 phosphosites have been described before [21 (link),23 (link)]. Other polyclonal antibodies used include those to Lck (Santa Cruz Biotechnology), GAPDH (Santa Cruz Biotechnology), PLCγ1 (Cell Signaling Technology), Slp76 (Cell Signaling Technology), p-PKD (Cell Signaling), p-Erk (Cell Signaling), Shp1 (Cell Signaling) and Shp2 (Abcam). Monoclonal antibodies utilized include those to phosphorylated tyrosine residues (Santa Cruz Biotechnology), EGFP (Covance), tubulin α (Santa Cruz Biotechnology), CD3 (UCHT1 clone, Merk Millipore), fluorescein-isothiocyanate-labeled CD45 (BD Biosciences), Zap70 (Upstate Biotechnology) and chicken IgM (Southern Biotech).

Barreira M., Rodríguez-Fdez S, & Bustelo X.R. (2018). New insights into the Vav1 activation cycle in lymphocytes. Cellular signalling, 45, 132-144.

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Cell Proliferation and Apoptosis Assay

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A cell counting kit (CCK) for WST assay was purchased from Donginbiotech Co. (Seoul, Korea). Crystal violet solution was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-phospho-AMPK, phospho-p38, phospho-ERK, phospho-JNK, cyclin D1, CDK4, AMPK, PARP, caspase-3, Bcl-2, Bcl-xL, and Bax antibodies were purchased from Cell Signaling (Danvers, MA, USA). Anti-p38, ERK, JNK, GAPDH, and α-tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Mun J.G., Jeon H.D., Yoon D.H., Lee Y.S., Park S.Y., Jin J.S., Park N.J, & Kee J.Y. (2022). Supercritical Extract of Cannabis sativa Inhibits Lung Metastasis in Colorectal Cancer Cells by Increasing AMPK and MAPKs-Mediated Apoptosis and Cell Cycle Arrest. Nutrients, 14(21), 4548.

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Comprehensive Protein Extraction and Analysis

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Protein extraction from cells and tissues and analysis via SDS-PAGE and Western blotting were carried out as described previously (Sha et al., 2009 (link); Yang et al., 2010 (link)). Quantification of signals was done using BioRad ImageLab software. Glycosylation status was assessed by incubation with EndoH and PNGaseF enzymes for 1 h at 37°C (NEB). Total protein levels were normalized with the help of loading controls. The following primary antibodies were used: KI67 (1:200, Abcam ab16667); CYCLIN D1 (1:100 for staining, 1:2000 for Western blot, Abcam ab16663); c-MYC (1:1000, MilliporeSigma C3956); V5 (1:5000 for Western blot, 1:250 for immunoprecipitation, Invitrogen R960-25); HA (1:3,000, Sigma H9658); WNT5A (1:2000 for Western blot, 1:100 for immunoprecipitation, Proteintech 55184-1-AP); WNT5B (1:1000, Abclonal A8313); WNT1 (1:1000, Proteintech 27935-1-AP); E-CADHERIN (1:1000, BD 610181); α-TUBULIN (1:2,000, Santa Cruz sc-5286); HSP90 (1:1,000, Abcam ab13492); CALNEXIN (1:5000, Enzo ADI-SPA-860-F); SEL1L (1:2,000, Abcam ab78298); HRD1 (1:300, kindly gifted by Dr. Richard Wojcikiewicz, SUNY Upstate Medical University for Western blot; 1:1000, Sigma HPA024300 for immunostaining of human liver tumors). The following secondary antibodies were used: goat anti-mouse and goat anti-rabbit IgG-HRP (1:5,000; BioRad).

Bhattacharya A., Wei J., Song W., Gao B., Tian C., Wu S.A., Wang J., Chen L., Fang D, & Qi L. (2022). SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer. iScience, 25(10), 105183.

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Western Blot Antibody Protocol

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Antibodies used in this study were against α-tubulin (Santa Cruz), pT389-p70S6K, p70S6K, ATF4 (Cell Signalling), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam). Horseradish peroxidase-conjugated donkey anti-mouse IgG and anti-rabbit IgG were used as Western blot secondary antibodies (Millipore).

van Geldermalsen M., Quek L.E., Turner N., Freidman N., Pang A., Guan Y.F., Krycer J.R., Ryan R., Wang Q, & Holst J. (2018). Benzylserine inhibits breast cancer cell growth by disrupting intracellular amino acid homeostasis and triggering amino acid response pathways. BMC Cancer, 18, 689.

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Antibody Sources for Protein Analysis

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Antibodies used in this study were obtained from the following sources: α-tubulin (Santa Cruz Biotechnology, CA, USA): Hemagglutinin (HA) (Roche); Erk, and phosphorylated Erk (Cell Signaling Technology, MA, USA). Recombinant SEMA3E was purchased from R&D Systems (R&D Systems, MN, USA).

Maejima R., Tamai K., Shiroki T., Yokoyama M., Shibuya R., Nakamura M., Yamaguchi K., Abue M., Oikawa T., Noguchi T., Miura K., Fujiya T., Sato I., Iijima K., Shimosegawa T., Tanaka N, & Satoh K. (2016). Enhanced expression of semaphorin 3E is involved in the gastric cancer development. International Journal of Oncology, 49(3), 887-894.

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Protein Isolation and Western Blotting

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The protein isolation and western blotting procedures have been described previously (19 (link)). Briefly, tissues were homogenized, the proteins separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotting performed using the following antibodies: eukaryotic elongation factor 2 (eEF2; total and phosphorylated Thr56; Cell Signaling Technology), eukaryotic initiation factor 2α (eIF2α; total and phosphorylated Ser51; Cell Signaling Technology), eIF4E binding protein 1 (4EBP1; total; Bethyl Laboratories, Inc.; phosphorylated Thr70; Cell Signaling Technology), ribosomal protein (rp) S6 kinase-1 (S6K1; total; Santa Cruz Biotechnology; phosphorylated Thr389; EMD Millipore). α-Tubulin (Santa Cruz Biotechnology) was used as a loading control, and phosphorylated proteins were normalized to their corresponding nonphosphorylated forms. An enhanced chemiluminescence kit (Catalog no. RPN 2232 and RPN 2235; GE Health Sciences) was used to visualize and analyze band intensity using a ChemiDoc-It Imaging System (Bio-Rad).

El-Kadi S.W., Boutry-Regard C., Suryawan A., Nguyen H.V., Kimball S.R., Fiorotto M.L, & Davis T.A. (2020). Intermittent Bolus Feeding Enhances Organ Growth More Than Continuous Feeding in a Neonatal Piglet Model. Current Developments in Nutrition, 4(12), nzaa170.

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Protein Abundance Detection Protocol

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Protein extractions were performed as previously described^{44 (link),49}. Primary antibodies HAS2 (Santa Cruz Biotechnology, sc-34068) (1:200 dilution), HSL (Santa Cruz Biotechnology, sc-74489) (1:200 dilution), α Tubulin (Santa Cruz Biotechnology, sc-53030) (1:200 dilution), Actin (Sigma #A4700) (1:1000 dilution), Adiponectin (homemade) (1:1000 dilution) were used. Protein abundance was detected using the one of the following secondary antibodies: Goat anti-Mouse IRDye 680RD (Li-cor 926-68070), Goat anti-rabbit IRDye 800CW (Li-cor 925-32211) or Goat anti-Rat DyLight 800 (Thermo Fisher SA5-10024) at 1:10,000 dilutions. Antibody decorated membranes were then visualized on a Li-Cor Odyssey infrared scanner (Li-Cor Bioscience). The scanned data were analyzed using Odyssey Version 3.0 software (Li-Cor Bioscience).

Zhu Y., Li N., Huang M., Bartels M., Dogné S., Zhao S., Chen X., Crewe C., Straub L., Vishvanath L., Zhang Z., Shao M., Yang Y., Gliniak C.M., Gordillo R., Smith G.I., Holland W.L., Gupta R.K., Dong B., Caron N., Xu Y., Akgul Y., Klein S, & Scherer P.E. (2021). Adipose tissue hyaluronan production improves systemic glucose homeostasis and primes adipocytes for CL 316,243-stimulated lipolysis. Nature Communications, 12, 4829.

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Western Blot Immunodetection Protocol

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Western blot (WB) was performed as described previously [42 (link)]. The primary antibodies used in this study were anti-SET7/9 (rabbit monoclonal, C24B1, 1:1000; Cell Signaling Technology, Danvers, MA; and mouse monoclonal, clone 5F2.3, 1:500; Merck Millipore), anti-SREK1IP1 (rabbit polyclonal, 1:200; GeneTex Inc, Irvine, CA), anti-H3K4me1 (No. 39298, rabbit polyclonal, 1:5000; Active Motif, Carlsbad, CA), anti-FLAG (mouse monoclonal M2, 1:10,000; Sigma-Aldrich Japan), and anti-α-Tubulin (mouse monoclonal, 1:400; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies.α-Tubulin was used as an internal control for WB.

Akiyama Y., Koda Y., Byeon S.J., Shimada S., Nishikawaji T., Sakamoto A., Chen Y., Kojima K., Kawano T., Eishi Y., Deng D., Kim W.H., Zhu W.G., Yuasa Y, & Tanaka S. (2015). Reduced expression of SET7/9, a histone mono-methyltransferase, is associated with gastric cancer progression. Oncotarget, 7(4), 3966-3983.

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Western Blot Analysis of Protein Extraction

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After treatment, protein was isolated by resuspending cells in M-PER^® Mammalian Protein Extraction Reagent (Thermo Scientific) and NE-PER lysate (Thermo Scientific) for immunoprecipitation with protease & phosphatase inhibitor cocktails (Thermo Scientific), incubated for 10 min at 4 °C, and centrifuged at 16,000 × g for 10 min to remove cell debris. Extracts were loaded onto 10% polyacrylamide gels and transferred to Hybond-ECL membranes (Amersham, Pollards Wood, UK). Membranes were blocked with 5% skim milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST). The membranes were then incubated with specific primary antibodies diluted with PBST at 4 °C with gentle shaking overnight: p65, ATF3, topoisomerase II-α, IκBζ, α-tubulin and β-actin (Santa Cruz Biotechnology); ac-p65 (Abcam) and HDAC1 (Millipore). After several washings, the membranes were incubated with anti-mouse or anti-rabbit immunoglobulin G peroxidase-conjugated antibody (Thermo Scientific) diluted in PBST (1:1,000) for 2 h. After the membranes were washed several times with PBST, detection was carried out using Pierce ECL Western blotting substrate (Thermo Scientific) and exposure of the blots to a LAS-1000 system (Fuji, Tokyo, Japan). The intensities of protein bands were measured using the ImageJ software (http://rsb.info.nih.gov/ij/)39 (link).

Kwon J.W., Kwon H.K., Shin H.J., Choi Y.M., Anwar M.A, & Choi S. (2015). Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-κB. Scientific Reports, 5, 14470.

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