Ab75783

Manufactured by Abcam

Ab75783 is a primary antibody product manufactured by Abcam. It is designed for use in common laboratory techniques.

Automatically generated - may contain errors

Lab products found in correlation

Ab124903, by Abcam (2 mentions) Ab229213, by Abcam (2 mentions) Chemidoc imager, by Bio-Rad (2 mentions) Novex nupage sds page system, by Thermo Fisher Scientific (2 mentions) Tank blot wet transfer, by Bio-Rad (2 mentions) Ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions) Tween 20, by Merck Group (2 mentions) Ab9485, by Abcam (2 mentions) Ab93779, by Abcam (1 mentions) T5192, by Merck Group (1 mentions) T9026, by Merck Group (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Volocity software, by PerkinElmer (1 mentions) 0.45 μm nitrocellulose membrane, by Bio-Rad (1 mentions) Anti mouse or anti rabbit secondary antibodies, by Bio-Rad (1 mentions) 0.45 m nitrocellulose membrane, by Bio-Rad (1 mentions)

3 protocols using ab75783

Immunofluorescence Imaging of Mitotic Spindle

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HeLa cells were cultured on ethanol-sterilized coverslips in 12-well plates and were synchronized or monastrol-treated before fixation with ice-cold methanol at −20 °C for 10 min, followed by rehydration with 1 × PBS at room temperature for 10 min. The fixed cells were permeabilized with 0.3% Triton X-100 in 1 × PBS for 15 min and blocked with 2% bovine serum albumin for 45 min at room temperature. Mouse anti-α-tubulin 1:2,000 (T9026, Sigma), rabbit anti-γ-tubulin 1:200 (T5192, Sigma), human anticentromere antibody 1:2,000 (HSM0101, ImmunoVision), rabbit anti-Kid 1:200 (ab75783, Abcam) and rabbit anti-NuSAP 1:200 (ab93779, Abcam) were utilized to stain the respective proteins in cells. Alexa Fluor dye-conjugated goat anti-mouse, goat anti-rabbit or goat anti-human IgG (Invitrogen) were used as the secondary antibodies. DNA was stained with Hoechst 33342 (Invitrogen). Cells were mounted in FluorSave reagent (Calbiochem). The images were processed using either the Volocity software (PerkinElmer) or Image J (National Institutes of Health, Bethesda, MD), as required.

Li C., Xue C., Yang Q., Low B.C, & Liou Y.C. (2016). NuSAP governs chromosome oscillation by facilitating the Kid-generated polar ejection force. Nature Communications, 7, 10597.

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Detailed SDS-PAGE Western Blotting Protocol

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Samples were separated by Novex NuPAGE SDS–PAGE system (Thermo Fisher Scientific) using 4–12% Bis-Tris in MES running buffer according to the manufacturer’s guidelines, and transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad) by tank-blot wet transfer (Bio-Rad) at room temperature for 1 h. Blocking, primary antibody incubations (4 °C, 16 h) and secondary antibody incubations (room temperature, 1.5 h) were performed in 5% (w/v) milk (Maresi, Fixmilch instant milk powder) in PBS, containing 0.05% Tween-20 (Sigma-Aldrich). SMC4 was probed using a rabbit polyclonal antibody (Abcam, ab229213, GR3228108-5, 1:1,000). GAPDH was probed with a rabbit polyclonal antibody (Abcam, ab9485, GR3212164-2, 1:2,500). hKid was probed with a monoclonal rabbit antibody (Abcam, ab75783, GR129278-4, 1:1,000). KIF4A was probed with a recombinant rabbit antibody (Abcam, ab124903, GR96215-7, 1:1000). Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, 1:10,000) were visualized using ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) on a Bio-Rad ChemiDoc Imager operated by analysed using Bio-Red Image Lab v.6.0.1, which was also used for analysis.

Schneider M.W., Gibson B.A., Otsuka S., Spicer M.F., Petrovic M., Blaukopf C., Langer C.C., Batty P., Nagaraju T., Doolittle L.K., Rosen M.K, & Gerlich D.W. (2022). A mitotic chromatin phase transition prevents perforation by microtubules. Nature, 609(7925), 183-190.

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Western Blotting for Protein Analysis

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Samples were separated by Novex NuPAGE SDS–PAGE system (Thermo Fisher Scientific) using 4–12% Bis-Tris in MES running buffer according to the manufacturer’s guidelines, and transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad) by tank-blot wet transfer (Bio-Rad) at room temperature for 1 h. Blocking, primary antibody incubations (4 °C, 16 h) and secondary antibody incubations (room temperature, 1.5 h) were performed in 5% (w/v) milk (Maresi, Fixmilch instant milk powder) in PBS, containing 0.05% Tween-20 (Sigma-Aldrich). SMC4 was probed using a rabbit polyclonal antibody (Abcam, ab229213, GR3228108-5, 1:1,000). GAPDH was probed with a rabbit polyclonal antibody (Abcam, ab9485, GR3212164-2, 1:2,500). hKid was probed with a monoclonal rabbit antibody (Abcam, ab75783, GR129278-4, 1:1,000). KIF4A was probed with a recombinant rabbit antibody (Abcam, ab124903, GR96215-7, 1:1000). Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, 1:10,000) were visualized using ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) on a Bio-Rad ChemiDoc Imager operated by analysed using Bio-Red Image Lab v.6.0.1, which was also used for analysis.

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