Acetylated tubulin

Manufactured by Santa Cruz Biotechnology

Sourced in Germany

Acetylated tubulin is a component of the cytoskeleton found in eukaryotic cells. It is a post-translationally modified form of the protein tubulin, where the lysine residue at position 40 is acetylated. Acetylated tubulin plays a role in the stabilization of microtubules, which are critical structures involved in cellular processes such as intracellular transport and cell division.

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Lab products found in correlation

Alexa 488, by Thermo Fisher Scientific (2 mentions) Dykddddk epitope tag antibody, by Novus Biologicals (2 mentions) Histone h3 pser10 antibody, by Novus Biologicals (2 mentions) Cyanine3 (cy3), by Jackson ImmunoResearch (2 mentions) Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions) Dmi6000b inverted microscope, by Leica (1 mentions) Alexa fluor 488 goat anti mouse a 11001, by Thermo Fisher Scientific (1 mentions) β catenin, by Cell Signaling Technology (1 mentions) Western blotting reagents, by Thermo Fisher Scientific (1 mentions) Gata1, by Abcam (1 mentions) Alpha tubulin, by Abcam (1 mentions) Chemidoc mp, by Bio-Rad (1 mentions) Prolong gold antifade mounting solution, by Thermo Fisher Scientific (1 mentions) Sqstm1 p62, by Cell Signaling Technology (1 mentions) Gabarapl1, by Proteintech (1 mentions) γ tubulin, by Merck Group (1 mentions) Gapdh, by Bio-Techne (1 mentions) Fluorchem 9900 imaging system, by Bio-Techne (1 mentions) Bis tris polyacrylamide gel, by Thermo Fisher Scientific (1 mentions) Gapdh, by Santa Cruz Biotechnology (1 mentions)

7 protocols using acetylated tubulin

Immunofluorescence Staining of Cell Markers

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MDA-MB-231 cells were plated on glass coverslips and treated as described. Cells were fixed in 10% formalin, blocked in 5% BSA in TBS containing 0.1% Triton-X (Roche), incubated in 1:500 primary antibody (β-catenin, Cell Signaling Technologies; Cat. No. 9581S), (PARD3 (ab64646)/ Numb (ab14140)/ APT1 (ab91606)/ GFP (ab290)/ Caveolin (ab17052), Abcam), (DHHC20, Sigma; Cat. No. HPA014483), (Acetylated tubulin, Santa Cruz; Cat. No. sc23950), 1:1000), for 1-2 hours at room temperature, incubated in secondary antibody (Alexafluor 488 goat anti-mouse (A11001)/ Alexafluor 594 goat anti-rabbit (A11012), Life Technologies; 1:1000) for 1 hour at room temperature, and mounted in DAPI-mount (Southern Biotech; Cat. No. 0100-20). ESCs were cultured without LIF, MEK or GSK inhibitors for 24 hours before staining with 1:1000 anti-GFP (ab290) as described. Cells were imaged using the Leica DMI6000 B inverted microscope on 40X magnification and colonies were imaged on 20X magnification.

Stypulkowski E., Asangani I.A, & Witze E.S. (2018). The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division. Science signaling, 11(511), eaam8705.

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Western Blotting Techniques for Protein Analysis

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Western blotting reagents were purchased from Thermo Fisher Scientific unless otherwise stated. Cells were lysed in M-Per mammalian protein extraction reagent (cat#78501) supplemented with Halt® protease and phosphatase inhibitor cocktail, following manufacturer’s instructions. Quantification of total protein was achieved by using a bicinchoninic acid (BCA) assay. For each sample, 20–40μg of protein were separated by electrophoresis using 12% Tris-Glycine gels. Protein transfers were performed using the iBlot2 system (20V for 1 minute, 23V for 3 minutes, and 25V for 2 minutes setting). PVDF membranes were blocked in 4% milk- Tris Buffered Saline with Tween 20 (TBST) buffer for 1 h, incubated in primary antibody overnight followed by the HRP secondary for 1 h.
The sources of primary antibodies used were: GATA-1 (Abcam; cat#89505, mouse monoclonal), acetylated tubulin (Santa Cruz, Dallas, TX; cat#sc-23950, mouse monoclonal), alpha tubulin (Abcam; cat#4074, rabbit polyclonal), histone H3 (ProSci, Poway, CA; cat#31007), and acetylated histone H3 (Thermo Fisher Scientific; Lys6, cat#MA5–11195 and Lys36, cat#MA-24672). Secondary HRP labeled, anti-mouse (cat#6728) and anti-rabbit (cat#6721) antibodies were purchase from Abcam. Membranes were incubated for 1 min in ECL reagent and captured using ChemiDoc MP (BioRad, Hercules, CA).

Simic D, & Sang N. (2019). Compounds targeting Class II histone deacetylases do not cause panHDACi associated impairment of megakaryocyte differentiation. Experimental hematology, 72, 36-46.

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Whole Mount Immunofluorescence of Embryos

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For whole mount immunofluorescence, embryos were fixed in 10% 10XMEMFA (0.1 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO₄, 3.7% formaldehyde), 10% formaldehyde and 80% water for 2 hours at room temperature and the vitelline envelope was removed manually. Embryos were permeabilized overnight in 1XPBS, 0.5% Triton, 1% DMSO (Perm solution) and blocked for 2 hours in 10% Normal Goat serum in Perm solution. Embryos were then incubated with primary antibodies. The primary antibodies used were: 40LoVe (CvH7, C579 kindly provided by Dr. Iain W. Mattaj, EMBL, Heidelberg, Germany), GFP (Invitrogen), DYKDDDDK Epitope Tag Antibody (Novus), Acetylated Tubulin (Santa Cruz) and Histone H3 [pSer10] Antibody (Novus). The incubation was performed overnight at 4°C. Embryos were then washed four times in Perm solution for 20 min, incubated for 2 hours RT with secondary antibodies. The secondary antibodies used were: Cy3 (Jackson Immunoresearch) and Alexa-488 (Invitrogen). Then the embryos washed four times in Perm solution for 20 min. Clearing of embryos was performed by immersing the embryos in 2∶1 BB: BA.

Andreou M., Yan C.Y, & Skourides P.A. (2014). 40LoVe and Samba Are Involved in Xenopus Neural Development and Functionally Distinct from hnRNP AB. PLoS ONE, 9(1), e85026.

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Ciliogenesis and Autophagy Imaging in RPE-1 and Jurkat Cells

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RPE-1 cells were seeded onto 12 mm coverslips (Life Technologies) and Jurkat cells were adhered on polysine slides for 15 min (VWR, 631-0107). For ciliogenesis and centriolar satellite staining, the cells were fixed in 4% paraformaldehyde for 12 min at room temperature, incubated with ice-cold methanol for 2 min, and quenched for 10 min with PBS/100 mM glycine. Fixed cells were incubated for 60 minutes with primary antibodies and 60 minutes with secondary antibodies in PBS 1X-0.2% BSA-0.05% Saponin. Coverslips were sealed with Prolong gold anti-fade mounting solution (Life Technologies), and nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The following primary antibodies were used: PCM1 (Cell signaling, 5213), γTubulin (Sigma, GTU88), and acetylated Tubulin (Santa Cruz Biotechnology, 6-11 B-1). To stain autophagy markers, cells were fixed in PBS-paraformaldehyde 4% and permeabilized with PBS 4%BSA 0.3% Triton X-100. The following primary antibodies were used: SQSTM1/p62 (Cell signaling, 88588) and GABARAPL1 (Proteintech, 11010-1-AP). Images were acquired on a Nikon A1 Rsi, using a 60× oil-immersion lens (Nikon Excellence Center, MicroPicell, SFR Francois Bonamy, Nantes, France). Images were processed using FIJI software.

Renaud C.C., Nicolau C.A., Maghe C., Trillet K., Jardine J., Escot S., David N., Gavard J, & Bidère N. (2024). Necrosulfonamide causes oxidation of PCM1 and impairs ciliogenesis and autophagy. iScience, 27(4), 109580.

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Acetylated Tubulin and Histone Detection

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Cell culture lysates were separated with electrophoresis on a 10% Bis-Tris polyacrylamide gel (Invitrogen, Carlsbad, CA), and the proteins were transferred to a nitrocellulose membrane. The membranes were probed with polyclonal antibodies for Acetylated tubulin (AcTub) and histones (AcHH3 and 4) as previously described [33 (link)]. Tubulin and glyceraldehyde 3-phosphate dehydrogenase (GADPH) were provided as controls. Acetylated tubulin, tubulin, acetylated-HH4, acetylated HH3, and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The density of the bands was analyzed and normalized against GAPDH control using the Alpha Innotech Fluorchem 9900 imaging system while running Alpha Ease FC software 3.3 (Alpha Innotech, San Leandro, CA).

Perron N.R., Nasarre C., Bandyopadhyay M., Beeson C.C, & Rohrer B. (2021). SAHA is neuroprotective in in vitro and in situ models of retinitis pigmentosa. Molecular Vision, 27, 151-160.

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Whole Mount Immunofluorescence Imaging

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For whole mount immunofluorescence, embryos were fixed in 10% 10XMEMFA (0.1 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO 4 , 3.7% formaldehyde), 10% formaldehyde and 80% water for 2 hours at room temperature and the vitelline envelope was removed manually. Embryos were permeabilized overnight in 1XPBS, 0.5% Triton, 1% DMSO (Perm solution) and blocked for 2 hours in 10% Normal Goat serum in Perm solution. Embryos were then incubated with primary antibodies. The primary antibodies used were: 40LoVe (CvH7, C579 kindly provided by Dr. Iain W. Mattaj, EMBL, Heidelberg, Germany), GFP (Invitrogen), DYKDDDDK Epitope Tag Antibody (Novus), Acetylated Tubulin (Santa Cruz) and Histone H3 [pSer10] Antibody (Novus). The incubation was performed overnight at 4uC. Embryos were then washed four times in Perm solution for 20 min, incubated for 2 hours RT with secondary antibodies. The secondary antibodies used were: Cy3 (Jackson Immunoresearch) and Alexa-488 (Invitrogen). Then the embryos washed four times in Perm solution for 20 min. Clearing of embryos was performed by immersing the embryos in 2:1 BB: BA.

Menezes A.C.S.C., Suzuki M.F., Oliveira J.E., Ribela M.T.C.P., Furigo I.C., Donato J J.r., Bartolini P, & Soares C.R.J. (2017). Expression, purification and characterization of the authentic form of human growth hormone receptor antagonist G120R-hGH obtained in Escherichia coli periplasmic space. Protein expression and purification, 131.

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Immunoblotting of Cellular Proteins

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Whole-cell lysates were prepared in RIPA buffer and analyzed by Western blotting. All blots were repeated at least twice. The antibodies used for Western blotting were: Anti-SKAP, MAP4, HA, Cbl, acetylated tubulin (Santa Cruz Biotech, Dallas, Tex), SKAP (HPA04207, Atlas Antibodies, Bromma, Sweden), FLAG-M2 (Sigma-Aldrich, St Louis, Mo), actin (Sigma-Aldrich), detyrosinated tubulin (ab48389, Abcam, Cambridge, United Kingdom).

Sharfe N., Karanxha A., Dadi H., Merico D., Chitayat D., Herbrick J.A., Freeman S., Grinstein S, & Roifman C.M. (2018). Dual loss of p110δ PI3-kinase and SKAP (KNSTRN) expression leads to combined immunodeficiency and multisystem syndromic features. The Journal of allergy and clinical immunology, 142(2).

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