Cells (2×104) were seeded into 4-well chamber slides (Millipore, cat. # PEZGS0416) and incubated overnight. They were then treated with a high dose (5 μM) of APIO-EE-9 for 2 h, then fixed with cold methanol at −20°C for 20 min followed by soaking in 1% PBS/BSA with 0.3% Tritonx-100 for 30 min at room temperature. Antibodies were diluted in 1% PBS-BSA. A mouse anti-human α-tubulin (1:400, Santa Cruz Biotechnology) antibody and 488-conjugated goat anti-mouse IgG secondary antibody were used to detect α- tubulin expression. A rabbit anti-human γ-tubulin (1:400, Santa Cruz Biotechnology) antibody and 568-conjugated goat anti-rabbit IgG secondary antibody were used to detect γ- tubulin expression. The nucleus was stained with DAPI.
Mouse anti human α tubulin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-human α-tubulin is a primary antibody that binds specifically to the α-tubulin subunit of microtubules in human cells. It can be used to detect and visualize the microtubule cytoskeleton in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680lt, by LI COR (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
3 protocols using mouse anti human α tubulin
1
Visualizing Microtubule Dynamics
2
Antibody Selection and Validation
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The monoclonal primary antibodies mouse anti-human CD74 (clone: By2) and mouse anti-human α-tubulin (clone: TU-02) were purchased from Santa Cruz Biotechnology, USA. Mouse antibody CD44 (clone: 156-3c11) was purchased from Cell Signaling Technology, USA. Rabbit anti-human β-actin (clone: poly 6221) was purchased from BioLegend, UK. The secondary antibody used for flow cytometry was a goat anti-mouse antibody conjugated with the fluorophore FITC (clone: poly4053) and was purchased from Bio-legend, UK. The secondary antibody used for Western blotting was either goat anti-mouse (IRDye 800CW) or goat anti-rabbit (IRDye 680 LT), purchased from LI-COR Biosciences. Finally, Alexa 488 (green) and Alexa 555 (red) antibodies were purchased from Life Technologies, UK.
3
Western Blot Analysis of TWIST1 Protein
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Cells were washed with PBS, and on ice, incubated in M-PER containing 1% Halt™ Protease Inhibitor Cocktail and 1% Halt™ Phosphatase Inhibitor (Thermo Scientific). Cell lysate was harvested and total protein content determined using a BCA assay (Thermo Scientific). For western blot analysis, 8 μg of total protein was electrophoresed on an 8-16 % SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with TBS-0.1 % Tween 20 (TBS-T) and 5-8 % bovine serum albumin (BSA) for 2 h, the membrane was probed with one of the following antibodies: mouse anti-human TWIST1 (1:400; Santa Cruz Biotechnology, CA, USA) or mouse anti-human α-tubulin (1:1000; Santa Cruz Biotechnology) in 5 % BSA. After washing with TBS-T, the membranes were incubated with peroxidase-conjugated anti-mouse antibody (1:2000; Dako, Glostrup, Denmark) in 2.5 % nonfat dried milk.
Quantification of blots was carried out using free ImageJ software with measurements recorded by 2 independent researchers.
Quantification of blots was carried out using free ImageJ software with measurements recorded by 2 independent researchers.
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