Anti γ tubulin t6557

Manufactured by Merck Group

Sourced in United States, Macao

Anti-γ-tubulin (T6557) is a primary antibody used in laboratory research. It specifically targets the γ-tubulin protein, which is a component of the microtubule-organizing center in eukaryotic cells. This antibody can be used in various immunoassay techniques to detect and study the localization and distribution of γ-tubulin within cells.

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Anti-tubulin (542 citations) γ-tubulin (169 citations) Anti-γ-tubulin (121 citations) T6557 (62 citations) Anti-tubulin (T6557) (3 citations) Anti-γ-tubulin antibody (T6557) (3 citations) Mouse anti-γ-tubulin (T6557) (2 citations)

12 protocols using anti γ tubulin t6557

Protein Purification and Immunoblotting Protocol

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Phenylmethylsulfonyl fluoride, sodium vanadate, bovine serum albumin, dithiothreitol (DTT), poly (Glu, Tyr), fluorescein isothiocyanate, holo-Transferrin, and buffer components were purchased from Sigma-Aldrich, St. Louis, MO, USA. Horseradish peroxidase linked anti-rabbit and anti-mouse secondary antibodies were from Cytiva, Marlborough, MA, USA. SuperSignal West Femto Chemiluminescent Substrate and Pierce ECL Western Blotting Substrate were purchased from Thermo Scientific, Waltham, MA, USA. Nickel-nitriloacetic acid (Ni-NTA) resin was from Qiagen, Germantown, MD, USA. The following antibodies and resin were obtained from MilliporeSigma, St. Louis, MO, USA: anti-phosphotyrosine clone 4G10 (05-321), anti-phospho-Ack1 (pY284) (09-142), anti-γ-Tubulin (T6557), horseradish peroxidase–linked anti-FLAG produced in mouse (F1804), and EZview red anti-FLAG M2 affinity resin. Anti-Mer antibody for Western blotting was from R&D Systems, Minneapolis, MN, USA (AF891). Anti-Mer antibody for immunoprecipitation was from abcam, Cambridge, UK (ab184086).

Hayashi S.Y., Craddock B.P, & Miller W.T. (2023). Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer. Kinases and phosphatases, 1(3), 167-180.

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Protein Purification and Immunoblotting Protocol

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Hayashi S.Y., Craddock B.P, & Miller W.T. (2023). Phosphorylation of Ack1 by the Receptor Tyrosine Kinase Mer. Kinases and phosphatases, 1(3), 167-180.

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Western Blot Analysis of Nuclear Proteins

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Cell extracts were prepared in RIPA buffer (Pierce) supplemented with Protease Inhibitor Cocktail (Sigma) and separated on 4–12% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blotting was performed as described previously (6 (link)). Anti-P54nrb (sc-376865) and anti-PSF (sc-374502) antibodies were purchased from Santa Cruz Biotechnology. Anti-PSPC1 (ab104238), anti-hnRNP K (ab32969), anti-TCP1-β (ab92746) and anti-FUS (ab23439) were purchased from Abcam. Anti-GAPDH (G8795) and anti-γ-Tubulin (T6557) were purchased from Sigma. Anti-phospho-Cyclin D1 (Thr286) (3300) was purchased from Cell Signaling.

Shen W., Liang X.H., Sun H, & Crooke S.T. (2015). 2′-Fluoro-modified phosphorothioate oligonucleotide can cause rapid degradation of P54nrb and PSF. Nucleic Acids Research, 43(9), 4569-4578.

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Immunoblotting and Immunoprecipitation Reagents

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Reagents and their suppliers were as follows: Chelex 100 resin from BioRad (142-2832; Mississauga, ON, Canada), cholera toxin (100) from List Biological (Campbell, CA, USA), leptomycin B (ALX-380-100) from Enzo Life Sciences (Plymouth, PA), PEI (25 kDa linear, Cat. No. 23966) from Polysciences (Warrington, PA). All other reagents were purchased from Sigma (St. Louis, MO, USA). The antibodies used were: anti-V5 (R960-25; Invitrogen, Carlsbad, CA), anti-HA (Y-11) from Santa Cruz (Santa Cruz, CA), anti-glyceraldehyde 3-phosphodehydrogenase (GAPDH; ADI CSA 335) from Enzo Life Sciences (Farmingdale, NY), anti-γ-tubulin (T6557) from Sigma (St. Louis, MO); anti-Cdh1 (DH01-DCS-266, ab3242, Abcam, Cambridge, UK), and anti-c-myc (SC-40, Santa Cruz). Horseradish peroxidase-conjugated goat anti-mouse (115–035-146) and anti-rabbit (111-035-144) antibodies were from Jackson Immuno Research Laboratories (West Grove, PA). Alexa Fluor®-conjugated goat anti-rabbit and goat anti-mouse IgG were from Molecular Probes/Thermo Fisher Scientific (Eugene, OR). λ phosphatase (PO753S) was from New England Biolabs (Ipswich, MI).

Singh R.K, & Dagnino L. (2016). CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes. Oncotarget, 8(3), 4977-4993.

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Protein Extraction and Western Blotting Protocol

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Attached and floating cells were collected and combined for the preparation of protein extracts. Soluble cellular proteins were obtained as described previously (Reynolds and Zhitkovich, 2007 (link)). For detection of histones, cellular proteins were solubilized by boiling cells for 10 min in a 2% SDS buffer (2% SDS, 50 mM Tris-HCl pH 6.8, 10% glycerol, 20 mM N-ethylmaleimide) supplemented with protease and phosphatase inhibitors (Thermo Scientific). Solutions were cooled to room temperature and centrifuged at 10000×g for 10 min to remove occasional debris. Proteins were separated by SDS-PAGE and electrotransferred to ImmunoBlot PVDF membranes. The following primary antibodies were used: anti-histone H3 phosphorylated at Ser10 (9701), anti-CHK1 phosphorylated at Ser317 (2344) and anti-p53 phosphorylated at Ser15 (9284) from Cell Signaling; anti-γ-tubulin (T6557) was from Sigma. Primary antibodies were typically used at 1:1000 dilutions except for anti-histone H3 antibodies that were diluted 1:5000. Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse IgG (12-349, Millipore; 1:5000 dilution) and goat anti-rabbit IgG (7074, Cell Signaling; 1:2000 dilution). Band intensities were quantified by ImageJ and normalized for loading.

Ortega-Atienza S., Green S.E, & Zhitkovich A. (2015). Proteasome activity is important for replication recovery, CHK1 phosphorylation and prevention of G2 arrest after low-dose formaldehyde. Toxicology and applied pharmacology, 286(2), 135-141.

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Cell Line Cultivation and Inhibitor Assay

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HEK293, MCF-7, and MDA-MB-468 (DMEM) were purchased from ATCC (Manassas, VA). HCT116 p53 −/− cell line was provided by Dr. Vogelstein B (Johns Hopkins University). ACHN, A498, A704 (DMEM) and HCC1937 (RPMI) cells were obtained from Korea cell line bank. Other Cell lines (UMRC2; C2, UMRC2/VHL; C2V), provided by Dr. Jung, YJ (Pusan National University). Cells were maintained in DMEM. All kinds of cell lines were maintained in liquid medium containing 10% FBS and 1% antibiotics at 37°C growth chamber. General chemical inhibitors including Adriamycin (324380) and Colcemid (234109) were purchased from Calbiochem. B02 (SML0364), Estrogen (250155), Fulvestrant (I4409), Taxol (T7402), Tamoxifen (T5648) and 4-OHT (H7904) were purchased from Sigma. Antibodies against GST (sc-138), Actin (sc-1616), ER-α (sc-8002), β-tubulin (sc-9104) and HA (sc-7392) were purchased from Santa Cruz. Anti-γ-tubulin (T6557) and Myc (M5546) were provided by Sigma, anti-pVHL Ab (2738) was obtained from Cell signaling. Rad51 (05–530), BRCA1 (07–434) were purchased from Milliopore.

Jung Y.S., Chun H.Y., Yoon M.H, & Park B.J. (2014). Elevated Estrogen Receptor-α in VHL-Deficient Condition Induces Microtubule Organizing Center Amplification via Disruption of BRCA1/Rad51 Interaction. Neoplasia (New York, N.Y.), 16(12), 1070-1081.

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Antibody Characterization for Cell Biology

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Anti-γ-tubulin (T6557; 1:500) and anti-FLAG (M2; F1804; WB: 1:1,000, IF: 1:500) mouse monoclonal antibodies were purchased from Sigma-Aldrich. Anti-α-tubulin (A11126; 1:100) mouse monoclonal antibody was from Invitrogen. Anti-GFP (GF090R; 1:200), anti-Lamp2 (GL2A7; 1:200) and anti-Ter119 (#553670; 1:200) rat monoclonal antibodies were obtained from Nacalai Tesque, Abcam and BD Biosciences, respectively. Anti-CEP135 (ab75005; 1:100), anti-Ulk1 (A7481; 1:1,000) and anti-Cep63 (06-1292; 1:200) polyclonal antibodies were purchased from Abcam, Sigma-Aldrich and Millipore, respectively. Anti-Cep152 (A302-480A; 1:200) and anti-CP110 (A301-343A; 1:200) polyclonal antibodies were from Bethyl. Anti-Beclin1 (PD017; 1:1,000) and anti-p62/SQSTM1 (PM045; WB: 1:5,000, IF: 1:500) polyclonal antibodies were from Medical & Biological Laboratories (MBL). Anti-CPAP (11517-1-AP; 1:200) and anti-CEP63 (16268-1-AP; 1:200) polyclonal antibodies were from Proteintech Group. 3-MA, Bafilomycin A1 and E64d were from Sigma-Aldrich, and other chemicals were purchased from Nacalai Tesque.

Watanabe Y., Honda S., Konishi A., Arakawa S., Murohashi M., Yamaguchi H., Torii S., Tanabe M., Tanaka S., Warabi E, & Shimizu S. (2016). Autophagy controls centrosome number by degrading Cep63. Nature Communications, 7, 13508.

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Antibody Analysis of CCDC6, USP7, AR

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For biochemical analysis the antibodies anti-CCDC6 (ab56353) Abcam, the anti-USP7 (A300-033A) Bethyl, anti-AR (sc-7305) Santa Cruz Biotechnology (CA, USA) and anti-γ-tubulin (T6557), SIGMA-Aldrich, Inc, were utilized. Secondary antibodies were from Biorad, California. For the immunohistochemical studies the antibodies anti-CCDC6, [(HPA-019051), Sigma-Aldrich, Co. LLC] and anti-USP7 [(HPA-015641), Sigma-Aldrich, Co. LLC] were utilized. The anti-AR (sc-7305) was from SCBT (CA, USA).

Morra F., Merolla F., Napolitano V., Ilardi G., Miro C., Paladino S., Staibano S., Cerrato A, & Celetti A. (2017). The combined effect of USP7 inhibitors and PARP inhibitors in hormone-sensitive and castration-resistant prostate cancer cells. Oncotarget, 8(19), 31815-31829.

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Comprehensive Reagent Sourcing for Cellular Assays

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Cholera toxin (100) and insulin (16634) were from List Biological (Campbell, CA, USA) and Invitrogen (Carlsbad, CA, USA), respectively. Polyethyleneimine (PEI; 25 kDa linear (23966) was from Polysciences (Warrington, PA). Chelex 100 resin was purchased from Biorad (142-2832; Mississauga, ON, Canada). All other reagents were purchased from Sigma (St. Louis, MO, USA). The antibodies used were: anti-V5 (R960-25; Invitrogen, Carlsbad, CA, or ab9113, Abcam, Cambridge, MA), and anti-E2F1 (TA308764; OriGene, Rockville, MD). Antibodies against cyclobutane pyrimidine dimers (CPD) were from CosmoBio Co. (NMDND001, clone TMD-2, Tokyo, Japan); anti-double strand (ds) DNA (ab27156, Abcam, Cambridge, MA); anti-HA (Y-11) and anti-Rad23A (D-6) antibodies were purchased from Santa Cruz (Santa Cruz, CA), anti-GAPDH was from Enzo Life Sciences (ADI CSA 335, Farmingdale, NY); anti-FLAG M2 (F1804) and anti-γ-tubulin (T6557) were from Sigma (St. Louis, MO). Horseradish peroxidase-conjugated goat anti-mouse (115–035-146) and anti-rabbit (111-035-144) antibodies were from Jackson Immuno Research Laboratories (West Grove, PA). Alexa Fluor®-conjugated phalloidin (A12379), goat anti-rabbit and goat anti-mouse IgG were from Molecular Probes/Invitrogen (Eugene, OR).

Singh R.K, & Dagnino L. (2016). E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair. Oncotarget, 7(18), 26275-26292.

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Immunofluorescence Assay for DNA Damage Response

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Primary antibodies: anti-γ-H2AX (613402 clone 2F3, Biolegend); anti-TRF1 (Karlseder lab); anti-TRF2 (Karlseder lab); anti-53BP1 (H-300) (sc-22760, Santa Cruz); anti-LIG4 (ab80514, Abcam); anti-pericentrin (ab4448, Abcam); anti-MPM-2 (05-368MG, Millipore); anti-γ-tubulin (T6557, Sigma-Aldrich); anti-H3S10P (D2C8) (3377, Cell Signaling); anti-GAPDH (A300-641A, Bethyl); anti-p53 (sc-126, Santa Cruz).
Secondary antibodies: HPR-linked anti-mouse or anti-rabbit (NXA931 or NA934V; GE Healthcare); Alexa-488-conjugated anti-rabbit (Invitrogen); Alexa-594-conjugated anti-mouse (Invitrogen).

Hayashi M.T., Cesare A.J., Rivera T, & Karlseder J. (2015). Cell Death During Crisis Is Mediated by Mitotic Telomere Deprotection. Nature, 522(7557), 492-496.

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