Rabbit anti-RIP3 antibody, Nec-1, and propidium iodide (PI) were obtained from Sigma-Aldrich (St Louis, MO, USA), rabbit anti-β-tubulin was from Abcam (Cambridge, UK), and the fluorescein isothiocyanate-Annexin V/PI apoptosis assay kit was from Clontech (Mountain View, CA, USA). Morpholino oligonucleotides were synthesized by Gene Tools, LLC (Philomath, OR, USA). Bicinchoninic acid assay was purchased from Pierce (Rockford, IL, USA). Lipid peroxidation (MDA) was obtained from Jian-Cheng Biotechnical Co. (Nanjing, Jiangsu, China). Goat anti-rabbit secondary antibody was obtained from Jackson Immuno Research Inc. (Lancaster, PA, USA).
Rabbit anti β tubulin
Manufactured by Abcam
Sourced in United Kingdom, United States
Rabbit anti-β-tubulin is a primary antibody that specifically recognizes β-tubulin, a major component of microtubules in eukaryotic cells. It can be used for the detection and quantification of β-tubulin in various applications such as Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti α tubulin, by Merck Group (5 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (2 mentions)
Lowry protein assay, by Bio-Rad (2 mentions)
Nupage bis tris 4 12 gradient polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Hrp conjugated anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Hrp conjugated anti mouse secondary antibody, by Jackson ImmunoResearch (2 mentions)
Mouse anti ha, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti guinea pig secondary antibodies, by Jackson ImmunoResearch (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Guinea pig anti htp 3, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Nec 1, by Merck Group (1 mentions)
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Morpholino oligonucleotide, by Gene Tools (1 mentions)
Rabbit anti rip3 antibody, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
19 protocols using rabbit anti β tubulin
1
Necroptosis Pathway Modulation Protocol
2
Immunofluorescence Staining of Cellular Proteins
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For immunofluorescence processing, cells were fixed with 4% Paraformaldehyde (Electron Microscopy Sciences) for 10 min followed by extraction with 0.3% Triton X-100 in PBS (Sigma-Aldrich) for 10 min. After blocking with 10% FBS in PBS with 0.1% Triton X-100, all primary antibodies were incubated at 4°C overnight. Then, the cells were washed with PBS containing 0.1% Triton X-100 and incubated with the respective secondary antibodies for 1 h at room temperature. Primary antibodies used were: mouse anti-Mad1 (1:500; Merck Millipore); mouse anti α-tubulin (1:2000; Sigma); rabbit anti-β-tubulin (1:2000; Abcam); anti-guinea pig CENP-C (1:1000; MBL International). Secondary antibodies used were Alexa Fluor 488, Alexa Fluor 568 and Alexa Fluor 647 (1:1000; Themofisher). DNA was counterstained with 1 μg/mL DAPI (4',6'-diamino-2-fenil-indol; Sigma-Aldrich) and mounted onto glass slides with 20 mM Tris pH8, 0.5 N-propyl gallate and 90% glycerol. Images were acquired using an AxioImager Z1 (63x, Plan oil differential interference contract objective lens, 1.46 NA; from Carl Zeiss), coupled with a CCD camera (ORCA-R2; Hamamatsu Photonics) and the Zen software (Carl Zeiss). Blind deconvolution of 3D image datasets was performed using Autoquant X software (Media Cybernetics).
3
Western Blot Analysis of Protein Targets
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Cell pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% Triton-X-100, 2 mM EDTA, 0.01% SDS, 50 mM NaF, 0.1 mM Na3VO4, 1×Protease Inhibitor/no EDTA), sonicated (20% amp for 15 s) and centrifuged at 15000 rpm for 15 min at 4 °C. 10 µg of cell lysate was denatured and resolved by 6% homemade SDS-PAGE protein gel [49 (link)]. Proteins were transferred onto nitrocellulose membranes and blocked with 10% skim milk 1× Tris-buffered saline, 0.1% Tween 20 (TBST). The membrane was probed with primary antibody; rabbit anti-AR (Santa Cruz, cat #sc-816), mouse anti-ER (Santa Cruz, cat #sc-8002), rabbit anti-PGR (Leica Biosystems, # NCL-L-PGR AB), rabbit anti-FOXA1 (Abcam, cat # ab23738), mouse anti-Myc (Sigma Aldrich, cat #M4439), mouse anti-V5 (Thermo Fisher, cat #46-0705), mouse anti-FLAG (Sigma Aldrich, cat #F3165) or rabbit anti-β-Tubulin (Abcam, cat #ab6046) in 1× TBST/2% skim milk overnight at 4°C. The next day, membranes were probed with anti-mouse immunoglobulins/HRP (Dako, cat #P0447) or goat anti-rabbit immunoglobulin/HRP (Dako, cat #P0448) in 1× TBST/2% skim milk, washed with 1× TBST and detected by Clarity Western enhanced chemiluminescence (ECL) substrate (Bio-Rad) using BioRad ChemiDoc MP Imaging System with Imagining Lab 5.0.
4
Quantitative Western Blot Analysis
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20 µg cell lysates were subjected to SDS–PAGE and electrically blotted on to PVDF membrane. After blocking with 2% (w/v) BSA, the blot was probed with rabbit anti-β-tubulin (Abcam), rabbit anti-actin (Abcam), rabbit anti-NMIIA (Abcam) or rabbit anti-S100P (R&D Systems). After extensive washing, anti-rabbit-HRP (Santa Cruz) 1/10,000 was used and the bands were visualised using ECL and their densities were measured using Image Lab software.
5
Immunofluorescence Staining of Muscle Cells
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Muscle sections, single myofibers, and cultured cells were fixed in 4% paraformaldehyde and permeabilized for 10 min in 0.3% Triton X-100 in PBS. The samples were blocked in blocking buffer (Beyotime Biotechnology) for 2 h at room temperature. Primary antibodies were incubated in blocking buffer at 4 °C overnight. Subsequently, the samples were washed with PBS and stained with the appropriate fluorescently labeled secondary antibodies (Alexa Fluor 350, 488, or 594) for 1 h at room temperature. After washing with PBS, DAPI (Roche) was used to stain nuclei for 3 min. The primary antibodies used were as follows: mouse anti-Pax7 (Developmental Studies Hybridoma Bank [DSHB], 1:50), mouse anti-eMyHC (DSHB, 1:50), mouse anti-MyoG (cat. no. ab1835, Abcam, 1:200), mouse anti-MyHC (cat. no. M4276, Sigma, 1:500), rabbit anti-laminin (cat. no. L9393, Sigma, 1:200), rabbit anti-Dvl2 (cat. no. ab22616, Abcam, 1:200), rabbit anti-β-tubulin (cat. no. ab6046, Abcam, 1:500), mouse anti-Flag (cat. no. F1804, Sigma, 1:500), and rabbit anti-HA (cat. no. ab9110, Abcam, 1:200). For live-cell staining, DiI (5 μM, Beyotime Biotechnology) and Hoechst 33342 (2 μg/ml, Beyotime Biotechnology) were applied for 20 min.
6
Evaluating the Effect of TetraV^V and Cisplatin on β-Tubulin Expression
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To assess the effect of TetraVV and cisplatin treatment on the expression of the microtubule’s subunit β-Tubulin, the drug-treated cells, and the control were washed twice with PBS and subjected to the lysis procedure using Radioimmunoprecipitation Assay (RIPA) buffer. All steps, including the sample preparation, electrophoretic separation, and immunological detection were performed as previously mentioned [6 (link)]. Primary antibodies: rabbit anti-β-Tubulin (1:1000, Abcam cat no. ab6046, Cambridge, UK) and mouse anti-β-actin (1:2000, Bio-Rad cat. no. MCA5775GA, Hercules, CA, USA) along with secondary antibodies conjugated with horseradish peroxidase (goat anti-mouse IgG and goat anti-rabbit IgG 1:10,000, Thermo Fisher Scientific cat no. 32430 and 32460, Waltham, MA, USA) were used for detection of target proteins. The protein expression of β-Tubulin was normalized to β-actin.
7
Hippocampus BDNF and NGF Protein Analysis
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Hippocampus tissues (n = 3 per group) were collected and lyzed by RIPA protein extraction buffer containing 1 × protease inhibitor. The total protein concentration was examined by BCA protein assay kit (Pierce, USA). Protein was separated by 10% SDS‐polyacrylamide gels and transferred to polyvinylidene fluoride membrane (Millipore, USA). Then the membranes were incubated within 5% nonfat milk for 1 h at RT, and then rabbit anti‐BDNF (1:1000, Abcam, UK) or rabbit anti‐NGF (1:1000, GeneTex, USA) antibody overnight at 4°C. Rabbit anti‐β‐Tubulin (1:5000, Abcam, UK) was applied as internal standard. The goat antirabbit IgG (Immunoglobulin G) conjugated with horseradish peroxidase (1:10,000; ab205719; Abcam; UK) was added to the membranes for 1 h at RT. At last, the blots were visualized and analyzed by Image J software (Bethesda, USA).
8
Western Blot Analysis of Protein Expression
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Western blot assays were performed as described previously (26 (link)). A total of 50 µg of prepared protein was loaded into each lane of SDS-PAGE gels. Gel electrophoresis was performed, and protein was transferred to a nitrocellulose membrane. The membrane was blocked in Carnation® non-fat milk and probed with primary and secondary antibodies. The following primary antibodies were used: rabbit anti-CB2R (1:500, Abcam, Cambridge, UK), mouse anti-CD68 (1:500, AbD Serotec, Oxford, UK), rabbit anti-CD206 (1:500, Santa Cruz Biotechnologies, Dallas, TX, USA), rabbit anti-phospho-CREB (Ser133; 1:1,000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-phospho-PKAC (Thr197; 1:1,000, Cell Signaling Technology, Boston, MA, USA), rabbit anti-β-tubulin (1:1,000, Abcam, Cambridge, UK), and mouse anti-β-actin (1:1,000, Santa Cruz Biotechnologies, Dallas, TX, USA). Then, membranes were incubated in the appropriate HRP-conjugated secondary antibody (diluted 1:1,000 in secondary antibody dilution buffer) for 1 h at 37°C. Protein bands were visualized using a nickel-intensified DAB solution, and the densitometric values were analyzed using Image J software. The housekeeping proteins β-tubulin and β-actin were used as internal controls.
9
Quantitative Western Blot Analysis of Cellular Proteins
10
Western Blot Analysis of COL17, β-Tubulin, and Integrin α6
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For Western blot analysis of whole-cell lysates, cells were lysed in RIPA buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma Aldrich), and the lysates were centrifuged. Each fraction was subjected to SDS–PAGE in 6% polyacrylamide gel. The gels were transferred onto nitrocellulose membranes. Blotting was performed using rabbit anti-COL17 (1:2,000 dilution) (26 (link)), rabbit anti-β-tubulin (Abcam, Tokyo, Japan, 1:20,000 dilution), and anti-integrin α6 (Santa Cruz, Dallas, TX, USA, 1:500 dilution) as the primary antibodies, followed by incubation with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Life Technologies, 1:5,000 dilution). Signals were visualized with Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA).
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