Samples were denatured in sample buffer and fractionated using SDS-PAGE gel. Then blots were transferred to polyvinylidene difluoride membranes (Immobilon-P, Merck Millipore), and reacted with mouse anti-FLAG M2 antibody (1:2000; Sigma, F1804), anti-V5 antibody (1:5000; abcam, ab27671), anti-tubulin antibody (1:2000; Sigma, T6199) or rabbit anti-histone H3 (1:2000; abcam, ab1791) for 1 h, subsequently with a horseradish peroxidase (HRP)-conjugated mouse IgG antibody (1:2000; Bio-Rad, immune-star anti-mouse HRP) or horseradish peroxidase (HRP)-conjugated rabbit IgG antibody (1:2000; Bio-Rad, immune-star anti-rabbit HRP) for 1 h. Clarity western ECL substrate (Bio-Rad) was used according to the manufacturer’s instructions to detect chemiluminescence. Fluorescent images were obtained using an ImageQuant LAS 4000 system (GE healthcare).
Anti tubulin antibody
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Anti-tubulin antibody is a laboratory reagent used in various research applications. It is a highly specific antibody that binds to tubulin, a protein found in the cytoskeleton of cells. This antibody can be used to detect and visualize the distribution of tubulin in cells, which is important for understanding cellular structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Rhodamine phalloidin, by Thermo Fisher Scientific (6 mentions)
Anti flag antibody, by Merck Group (5 mentions)
Clarity western ecl substrate, by Bio-Rad (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Protease and phosphatase inhibitor, by Roche (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Anti ha antibody, by Santa Cruz Biotechnology (3 mentions)
Mg132, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
T9026, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (2 mentions)
P gp mdr1, by Cell Signaling Technology (2 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Cleaved caspase 8, by Cell Signaling Technology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Bm chemiluminescence western blotting kit, by Roche (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
103 protocols using anti tubulin antibody
1
Western Blot Analysis of Protein Targets
2
E2F8 Regulation of Cell Cycle and Growth
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RT‐PCRs were performed using specific primers for E2F8 and β‐actin 32 , 33 . Antibody against E2F8 was obtained from Bethyl laboratories (Montgomery, TX). Antibodies to pCdk2Y15, pHistone H3S10, Cdk2, and Histone H3 were purchased from Abcam (Cambridge, UK). Anti‐tubulin antibody was supplied by Sigma‐Aldrich (St. Louis, MO). The cell extracts were resolved on 6–13.5% SDS‐PAGE gels and were probed with the indicated antibodies. Cell cycle and growth was assessed by propidium iodide staining and 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assays 15 . Mitotic block experiments were performed as previously described 34 . Cells were synchronized by arresting them at G1/S phase with 2 mmol/L thymidine 18 h, followed by a 4 h release, and then cells were arrested at G2/M phase with 100 ng/mL nocodazole for 16 h. Geraniol was treated at the same time as nocodazole. The cells were released from nocodazole block by washing with fresh medium. For a rescue experiment, PC‐3 cells were transfected with human E2F8 in pcDNA3 plus pEGFP and then incubated with 1 mmol/L geraniol for 24 h prior to flow cytometric analysis or western blotting.
3
Western Blot Analysis of Cell Signaling
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Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).
4
Western Blot Analysis of Oncogenic Pathways
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Recombinant human OSM (hOSM) was purchased from Stem cell technologies Inc (Grapevine, TX, USA). Antibody to p-Y705-STAT-3 was purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to STAT-3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-tubulin antibody was purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA). Human ADM peptide 1-52 and ADM fragment 22-52 were purchased from Sigma-Aldrich, Co. (St. Louis, MO, USA).
5
Visualizing V. cholerae Infection in Host Cells
6
Visualization of Cytoskeleton Dynamics
7
Whole-Cell Lysate Preparation and Immunoblotting
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Whole-cell lysates were prepared by washing cells directly in wells with ice-cold PBS, followed by lysis on ice in RIPA buffer (50 mM Tris-HCl/pH 7.5, 1% (wt/vol) Triton X-100, 1% (wt/vol) glycerol, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulphate, 137 mM sodium chloride, 1 mM sodium orthovanadate, and 0.5 mM EDTA) complemented with Complete™ protease inhibitor cocktail. Cell debris was removed by centrifugation (21,000 g, 10 min, 4°C) and the protein concentration of the supernatant was measured with BCA protein assay kit (Uptima, Montluçon, France).
LSR was detected with a rabbit polyclonal anti-LSR antibody (clone X-25) (dilution 1:500; sc-133765; Santa Cruz) and tubulin with a mouse monoclonal anti-tubulin antibody (dilution 1:10000; T9026; Sigma-Aldrich). Horseradish peroxidase-conjugated donkey anti-mouse IgG (H&L) (#610–703–124; Rockland) or goat anti-rabbit IgG (H&L) (#7074; Cell Signaling) antibodies (dilutions 1:3000) were used as secondary antibodies. Antibody signals were developed by enhanced chemiluminescence reaction.
LSR was detected with a rabbit polyclonal anti-LSR antibody (clone X-25) (dilution 1:500; sc-133765; Santa Cruz) and tubulin with a mouse monoclonal anti-tubulin antibody (dilution 1:10000; T9026; Sigma-Aldrich). Horseradish peroxidase-conjugated donkey anti-mouse IgG (H&L) (#610–703–124; Rockland) or goat anti-rabbit IgG (H&L) (#7074; Cell Signaling) antibodies (dilutions 1:3000) were used as secondary antibodies. Antibody signals were developed by enhanced chemiluminescence reaction.
8
Purification and Detection of Cell Surface Proteins
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Cell surface proteins were purified from transiently transfected HEK293T cells by use of the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, cells at 90-95% confluence in a T75 flasks were incubated with 10 ml of sulfo-NHS-biotin solutions (0.25 mg/ml in PBS) for 30 min at 4°C. After the reaction was quenched by 500 µl of quenching solution, cells were scraped, washed with TBS buffer and sonicated. The protein concentration was determined by Bradford Assay. Biotinylated proteins were incubated with Immobilized NeutrAvidin Gel (Pierce), eluted according to the manufacturer's directions and loaded onto SDS-PAGE, and immunoblots were probed with anti-GFP antibodies (1:1000, Roche, 11814460001) to detect GFP-VANGL2. Detection of tubulin with anti-tubulin antibody (1:2500, Sigma-Aldrich, T9026) was used as a threshold above which samples were considered to be contaminated with intracellular proteins. All experiments were done in biological triplicate.
9
Western Blot Analysis of EPC Proteins
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Western blot analysis was performed according to the manufacturer's instructions (Pierce). Briefly, transfected EPCs were lysed and harvested in cell lysis buffer with protease and phosphatase inhibitors (Roche). Protein concentration of the samples was determined using the Pierce BCA Protein Assay Kit (Invitrogen) and the samples were then boiled for 10 minutes. Lysates from EPCs containing 50µg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking by incubation in 5% non-fat dry milk for 1 hour at room temperature, the membranes were incubated with 1∶1000 dilution of primary rabbit antibodies against FOXO3a, p27kip1 and CDK2, and primary mouse antibodies against PCNA and cyclin D1 (Cell Signaling Technology) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology) at 1∶1000 dilutions for 1 hour. The protein bands were detected by ECL detection reagents (Millipore, Billerica, MA) and quantified with Quantity One System (Bio-Rad). Anti-tubulin antibody (Sigma, MO, USA) was used as a loading control.
10
Detecting Prestin Variants in HEK293T Cells
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Two days after transfecting HEK293T cells with either EGFP-gPrestin WT or with EGFP-gPrestin completely lacking exons 17 and 18 (ΔEx17-18), lysates were made by incubating the cell pellet in lysis buffer (50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% Triton X-100) supplemented with 1 mM PMSF and 1X proteinase inhibitor cocktail (Sigma) followed by centrifugation. The supernatant was then boiled in SDS sample buffer before loading the hand-cast 8% gel and subsequent transfer to nitrocellulose membranes. For the detection of EGFP-prestin variants, Living Colors® A. v. Monoclonal Antibody (JL-8) (Takara Bio USA; AB 10013427) was used. An anti-tubulin antibody (Sigma; AB 477583) was chosen as the loading control. Images were captured using the Kodak Gel Logic 2200 Imaging System controlled by Carestream MI software (Molecular Bioimaging, Bend OR).
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