Western blot analysis was carried out with rabbit polyclonal FVII antibody (Novus Biologicals) at 1:1000 dilution using 50 µg of T-47D cell lysate from each DHT-treated and control experiments. Membrane was stripped and immunoblotting with rabbit α-tubulin antibody (Abcam, Cambridge, UK) was applied to assess loading. To extract protein from the conditioned media, cell lines were cultured for 48 hours in serum-free media containing either DHT at 100 nM or control vehicle followed by concentration using Amicon Ultra-15 (3K) centrifugal filters (Millipore, Billerica, MA, USA). A total of 100 µg from each conditioned medium was precipitated and used for immunoblotting. Protein concentrations were measured using the BCA Protein Assay Kit (Fisher scientific). Immunoblot imaging and analysis of band densities were performed using ChemiDoc XRS system and Image Lab software, respectively (Bio-Rad, Hercules, CA, USA). Western blots were performed in three replicates and the average fold change was shown.
Rabbit α tubulin antibody
Manufactured by Abcam
Sourced in United Kingdom
Rabbit α-tubulin antibody is a primary antibody that specifically recognizes the α-tubulin protein, a major component of microtubules. It can be used for the detection and analysis of α-tubulin in various applications, such as Western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Fvii antibody, by Novus Biologicals (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Evos sl digital inverted microscope, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg h l cy3 preadsorbed, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
Pierce enhanced chemilumescent ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg antibody, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Goat anti rabbit igg, by Bio-Rad (1 mentions)
3 protocols using rabbit α tubulin antibody
1
Quantifying FVII Protein Expression
2
Immunofluorescence Analysis of Microtubules and PLK1
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CCRF-CEM cells were treated with 30 µM of compound (1) for 24 h. Then, the cells were washed with PBS and fixed with 3.7% paraformaldehyde for 30 min at room temperature. The cells were blocked for 1 h at room temperature using a blocking buffer (5% FBS and 0.3% Triton X-100 in PBS). Primary antibodies [rabbit α-tubulin antibody (Abcam, Cambridge, UK) and mouse PLK1 monoclonal antibody (Thermo Fisher Scientific)] were added and allowed to stand for 2 h at room temperature (dilution 1:1000). Then, the cells were washed with PBS three times. Secondary antibodies [goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam) or goat anti-mouse IgG H&L (Cy3®) preadsorbed (Abcam)] were subsequently added (dilution 1:1000). Then, the cells were washed with PBS, stained using 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich), and mounted using Fluoromount-G® (Southern Biotech, Birmingham, AL, USA). Fluorescence imaging was performed using an EVOS SL digital inverted microscope (Life Technologies).
3
Quantifying Apoptosis in 4T1 Cells
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4T1 cells were seeded at 1x106 cells per 35 mm dish. Next day, the cells were infected for 1 h at the indicated MOI of Ad virus. Following a 48 or 72 h incubation, whole-cell lysates were collected using 2 × SDS/PAGE protein loading buffer (62.5 mm Tris HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% β-mercaptoethanol). Samples were boiled for 5 min, separated by electrophoresis on a 15% SDS-polyacrylamide gel, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Etobicoke, ON, Canada). The resulting membrane was probed with a mouse HA tag monoclonal antibody (1:10 000, Cell Signaling (Beverly, MA, USA) #2367), rabbit cleaved caspase-3 monoclonal antibody (1:1000, Cell Signaling #9664) or full-length caspase-3 antibody (1:1000, Cell Signaling #9662) and 1:5000 goat anti-rabbit IgG (Bio-Rad, Mississauga, ON, Canada, #170-6515) or goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170-6516). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit α-tubulin antibody, AbCam (Toronto, ON, Canada) #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170-6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).
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