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Mouse anti tubulin e7

Manufactured by Developmental Studies Hybridoma Bank
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Mouse anti-tubulin (E7) is a monoclonal antibody that specifically binds to the alpha-tubulin subunit of microtubules. It is a useful tool for the detection and localization of microtubules in a variety of cell types and applications.

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Lab products found in correlation

Ripa buffer, by Thermo Fisher Scientific (4 mentions) Rat anti ha 3f10, by Roche (2 mentions) Bis tris gradient gel, by Thermo Fisher Scientific (2 mentions) Rabbit anti pchk1 s317, by Cell Signaling Technology (2 mentions) Rabbit anti pchk2 t68, by Cell Signaling Technology (2 mentions) Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions) Mouse anti myc, by Santa Cruz Biotechnology (1 mentions) Rabbit anti flag, by Merck Group (1 mentions) Rabbit anti myc, by Santa Cruz Biotechnology (1 mentions) Anti flag m2 affinity agarose gel, by Merck Group (1 mentions) Mouse anti flag, by Merck Group (1 mentions) Protein a sepharose, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Gfp trap agarose, by Proteintech (1 mentions) Protease inhibitor cocktail with edta, by Roche (1 mentions) Mouse anti flag m2, by Merck Group (1 mentions) Mouse anti myc 9e10, by Santa Cruz Biotechnology (1 mentions) Mouse anti v5 r960 25, by Thermo Fisher Scientific (1 mentions) Rabbit anti ha, by Cell Signaling Technology (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions)

Close spelling variants of this product

Mouse anti-β-tubulin E7 Mouse anti-beta-tubulin E7 E7 mouse monoclonal anti-β-tubulin Anti-tubulin (E7) Mouse anti-tubulin Anti-tubulin

6 protocols using mouse anti tubulin e7

1

Immunoprecipitation Assays for Protein Complexes

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For immunoprecipitation assays, cells were lysed in Lysis Buffer (50 mM Tris pH8, 150 mM NaCl, 1% NP-40, and 1 mM EGTA) supplemented with 0.1M NaF, phosphatase inhibitor cocktails 2 and 3 (Sigma) and protease inhibitor cocktail (Roche). Cell extracts were cleared at 16,000 g for 20 min at 4°C. FLAG-tagged proteins were purified using anti-FLAG M2 Affinity Agarose Gel (Sigma), whereas immunoprecipitation of Myc-tagged proteins was carried out using Protein A Sepharose (Sigma). Detection of purified proteins and associated complexes was performed by immunoblot analysis using chemiluminescence (Pierce). Western Blots were probed with mouse anti-FLAG (M2, Sigma), rabbit anti-FLAG (Sigma), mouse anti-Myc (9E10, Santa Cruz Biotechnology), rabbit anti-Myc (Santa Cruz Biotechnology), rat anti-HA (3F10, Roche Applied Science), mouse anti-Tubulin (E7, Developmental Studies Hybridoma Bank, DSHB) and mouse anti-GFP (in-house produced), rat anti-Yki [47 (link)], and rabbit [48 (link)] and guinea pig anti Sav (a kind gift from G. Halder).
Aerne B.L., Gailite I., Sims D, & Tapon N. (2015). Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4. PLoS ONE, 10(6), e0131113.
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2

Western Blot Analysis of Cell Signaling

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Cells were harvested with RIPA buffer (Cold Springs Harbor protocols) containing protease and phosphatase inhibitors, and proteins were separated on a 4%–12% Bis-Tris gradient gels (Thermo-Fisher). and transferred to Pure Nitrocellulose Blotting Membrane (Life Sciences) or Amersham™ Hybond™ PVDF membrane (0.45 μM GE Healthcare Life Science). Membranes were blocked with 4% BSA, incubated with primary antibody overnight (1:1000–1:2000 dilution), washed, and incubated with secondary fluorescent conjugate anti-bodies or HRP, followed by a last wash. Membranes were scanned using LI-COR Odyssey CLX, Bio-rad Chemidoc™ or exposed to film.
Primary antibodies used are: Mouse monoclonal anti-p53 (Santa Cruz Biotechnology Cat# sc-126, RRID:AB_628082), mouse monoclonal anti-MDM2 (Ab-1, Millipore Cat# OP46T-10UG, RRID:AB_564805), rabbit anti-pChk2 (T68) (Cell Signaling Technology), rabbit anti-pChk1 (S317) (Cell Signaling Technology), mouse anti-tubulin (E7) (Developmental Studies Hybridoma Bank), mouse monoclonal anti-β-Actin (AC-74) (Sigma-Aldrich Cat# A2228, RRID:AB_476697). Info on Secondary antibodies?
Tsabar M., Mock C.S., Venkatachalam V., Reyes J., Karhohs K.W., Oliver T.G., Regev A., Jambhekar A, & Lahav G. (2020). A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest. Cell reports, 32(5), 107995.
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3

Western Blot Analysis of Cell Signaling

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Cells were harvested with RIPA buffer (Cold Springs Harbor protocols) containing protease and phosphatase inhibitors, and proteins were separated on a 4%–12% Bis-Tris gradient gels (Thermo-Fisher). and transferred to Pure Nitrocellulose Blotting Membrane (Life Sciences) or Amersham™ Hybond™ PVDF membrane (0.45 μM GE Healthcare Life Science). Membranes were blocked with 4% BSA, incubated with primary antibody overnight (1:1000–1:2000 dilution), washed, and incubated with secondary fluorescent conjugate anti-bodies or HRP, followed by a last wash. Membranes were scanned using LI-COR Odyssey CLX, Bio-rad Chemidoc™ or exposed to film.
Primary antibodies used are: Mouse monoclonal anti-p53 (Santa Cruz Biotechnology Cat# sc-126, RRID:AB_628082), mouse monoclonal anti-MDM2 (Ab-1, Millipore Cat# OP46T-10UG, RRID:AB_564805), rabbit anti-pChk2 (T68) (Cell Signaling Technology), rabbit anti-pChk1 (S317) (Cell Signaling Technology), mouse anti-tubulin (E7) (Developmental Studies Hybridoma Bank), mouse monoclonal anti-β-Actin (AC-74) (Sigma-Aldrich Cat# A2228, RRID:AB_476697). Info on Secondary antibodies?
Tsabar M., Mock C.S., Venkatachalam V., Reyes J., Karhohs K.W., Oliver T.G., Regev A., Jambhekar A, & Lahav G. (2020). A Switch in p53 Dynamics Marks Cells That Escape from DSB-Induced Cell Cycle Arrest. Cell reports, 32(5), 107995.
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4

Co-Immunoprecipitation Assay Protocol

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S2 and HEK293T cells were lysed with HEPES lysis buffer (150 mM NaCl, 50 mM HEPES pH 7.5, 0.5% (v/v) Triton X-100 supplemented with protease inhibitor cocktail with EDTA (Roche) and the soluble fraction was obtained by centrifugation (16,000 × g, 20 min). For co-IP experiments cleared lysates were added to 20 μL of ANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) or 15 μL of GFP-Trap agarose (ChromoTek) in 100 μL HEPES lysis buffer. The lysate and the beads were incubated for 1 h at 4 °C and washed four times with HEPES lysis buffer. Input and co-IP samples were analysed by SDS-PAGE and western blot. Primary antibodies were: mouse anti-FLAG (M2), 1/5000 (Sigma-Aldrich F1804); mouse anti-GFP (3E1), 1/1000 (Cancer Research UK); rat anti-HA (3F10), 1/2000 (Roche); mouse anti-Myc (9E10), 1/5000 (Santa Cruz sc:40); mouse anti-Tubulin (E7), 1/5000 (Developmental Studies Hybridoma Bank); mouse anti-V5 (R960-25), 1/5000 (Thermo Fisher), rabbit anti-P-TAZSer89 1/1000 (E1X9C) (Cell Signalling Technologies 59971), rabbit anti-HA 1/1000 (C29F4) (Cell Signalling Technologies 3724). Secondary antibodies were from GE Healthcare (1:5000).
Bertran M.T., Mouilleron S., Zhou Y., Bajaj R., Uliana F., Kumar G.S., van Drogen A., Lee R., Banerjee J.J., Hauri S., O’Reilly N., Gstaiger M., Page R., Peti W, & Tapon N. (2019). ASPP proteins discriminate between PP1 catalytic subunits through their SH3 domain and the PP1 C-tail. Nature Communications, 10, 771.
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5

Western Blot Analysis of Apoptosis Markers

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Ten µg of protein from cell lysates were run on PAGE gels as we have done before[14 (link)] and transferred to a nitrocellulose membrane. The membrane was treated with blocking buffer for 1 h at room temperature, followed by incubation in 1:1000 dilution rabbit anti-cleaved Caspase-3 (Cell Signaling #9664), 1:2000 dilution rabbit anti-ISG54 (Thermo-Fisher #PA3-845), 1:1000 dilution of rabbit anti- p27Kip (Cell Signaling 3688). or 1:500 mouse anti-tubulin E7 (Developmental Studies Hybridoma Bank, University of Iowa, Department of Biological Sciences, Iowa City, IA) and then goat anti-mouse-IgG-Alexa Fluor680- (1/5000) or donkey anti-rabbit-IgG-IRDye® 800CW. The membrane was washed three times and then scanned with a LICOR Odyssey® Infrared Imaging System.
Guinn Z., Brown D.M, & Petro T.M. (2017). Activation of IRF3 contributes to IFN-γ and ISG54 expression during the immune responses to B16F10 tumor growth. International immunopharmacology, 50, 121-129.
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6

Quantitative Western Blot Analysis

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Antibodies used for Western blotting were diluted in PBS + 2.5% milk. Primary rabbit antibody to full-length Xenopus H1M (Maresca et al., 2005 (link)) was used 1:1,500. Primary antibody to histone H2A (rabbit, ab13923; Abcam) was used 1:1,000. Nap1 antisera were used at 1:4,000. Primary antibody to β-tubulin (mouse anti–tubulin E7; Developmental Studies Hybridoma Bank) was used at 1:5,000. Primary antibodies for γ-glutamylation, called TTβIII-glu and TTSG1 (rabbit, created against short synthetic glutamylated peptides derived from sequences from rat brain tubulin) were used at 1:10,000 as described in Spano and Frankfurter (2010) (link). TTβIII-glu and TTSG1 antibodies were provided by D. Shechter (Albert Einstein University, Bronx, NY). Secondary antibodies from Rockland Immunochemicals (goat anti–rabbit IRDye 800 or goat anti–mouse IRDye 700) were used at 1:10,000. Blots were scanned with an Odyssey Infrared Imaging System (LI-COR Biosciences), and band intensity was quantified with ImageJ.
Miller K.E, & Heald R. (2015). Glutamylation of Nap1 modulates histone H1 dynamics and chromosome condensation in Xenopus. The Journal of Cell Biology, 209(2), 211-220.
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