Proteins were separated using 10% gel with an electrophoresis cells (GE Life Technologies). To separate α- and β-tubulins, 10% gel was prepared using a stock solution of 40% acrylamide solution (Meryer) supplemented with 0.54% bisacrylamide (w/v) powder (Yuanye) according to a protocol reported previously (39 (link)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using a semidry blotter (GE Healthcare). Membranes were incubated with anti-His tag (1:5000 dilution; Immuno Way), anti-glutamylation (GT335, 1:4000 dilution; Adipogen), anti-polyglutamylation (polyE, 1:4000 dilution; Adipogen), anti-α-tubulin (EP1332Y, 1:4000 dilution; Abcam or DM1A, 1:4000 dilution; Sigma), or anti-β-tubulin (anti-β-tubulin, 1:50,000 dilution; Proteintech) antibody. Immunoreactivity of proteins was visualized with WesternBright ECL (Advansta) using Western Blot Imager (Vilber) after incubation with horseradish peroxidase–labeled goat antimouse (1:5000 dilution, Bioss) or donkey anti-rabbit (1:5000 dilution, Bioss) antisera.
Ep1332y
Manufactured by Abcam
Sourced in United States
EP1332Y is a primary antibody that targets the histone H3 (phospho S10) protein. It is a rabbit monoclonal antibody that can be used for various applications, including Western blotting and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico chemiluminescence substrate, by Thermo Fisher Scientific (2 mentions)
Criterion blotter, by Bio-Rad (2 mentions)
Donkey anti rabbit igg, by GE Healthcare (2 mentions)
Hrp labeled sheep anti mouse, by GE Healthcare (2 mentions)
Westernbright ecl, by Advansta (1 mentions)
Western blot imager, by Vilber (1 mentions)
Horseradish peroxidase labeled goat antimouse, by Bioss Antibodies (1 mentions)
Anti β tubulin, by Proteintech (1 mentions)
Anti gapdh antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Alexa fluor 488 labeled goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 555 labeled donkey anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Rabbit polyclonal anti his tag, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ab150083, by Abcam (1 mentions)
Immun blot, by Bio-Rad (1 mentions)
Mouse anti myc, by Takara Bio (1 mentions)
Gt335, by Adipogen (1 mentions)
8 protocols using ep1332y
1
Separation and Detection of Tubulin Isoforms
2
Antibody Panel for RSV and Tubulin
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The mouse polyclonal antibody against the RSV CP peptide was produced by HuaAn Biotechnology Co., Ltd (HuaBio, Hangzhou, China). The rabbit anti-RSV NS3 was kindly provided by Dr. Kun Zhang (Yangzhou University). Due to highly conserved α-2-tubulin, the rabbit monoclonal anti-α-TUB antibody (EP1332Y, Abcam, UK) was used to detect LsTUB in SBPHs. The following antibodies were obtained from the sources indicated: goat anti-mouse IgG HRP conjugate (cat. CW0102S, Cwbiotech, China), goat anti-rabbit IgG HRP conjugate (cat. CW0103S, Cwbiotech, China), Alexa Fluor 488-labeled goat anti-mouse IgG (cat. 115-545-003, Jackson ImmunoResearch Laboratories, USA), Alexa Fluor 555-labeled donkey anti-rabbit IgG (cat. ab150074, Abcam, UK), rabbit polyclonal anti-His tag (cat. 2365, Cell Signaling Technology, USA), and rabbit polyclonal anti-GAPDH antibody (cat. ab157156, Abcam, UK). DAPI (4’,6-diamidino-2-phenylindole) was from Sigma (cat. 28718-90-3, Sigma, USA).
3
Assessing Biocompatibility of Plasmonic NP-Graphene
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To examine the biocompatibility of the plasmonic NP-graphene interface, cells incubated for 24 h were fixed with 4% formaldehyde in PBS for 15 min at RT and permeabilized with 0.4% Triton X-100 for 15 min. The cells were washed twice with PBS for 15 min. For imaging microtubules, after blocking the cells with 5% BSA in PBS for 1 h, cells were incubated with a rabbit monoclonal anti-α-tubulin antibody (EP1332Y; Abcam, Cambridge, MA, USA) overnight at 4 ℃ and washed five times with PBS for 2 min over 5 times. The cells were further incubated with an Alexa 647-conjugated anti-rabbit secondary antibody (ab150083; Abcam, Cambridge, MA, USA) for 1 h at RT. F-actin was stained with Alexa Fluor 488-phalloidin (Invitrogen) for 1 h at RT. Nucleic acids were stained with the DNA-binding dye Hoechst 33258 (94403; Sigma-Aldrich). Stained cells were visualized using a confocal laser scanning microscope (LSM 800; Carl Zeiss, Jena, Germany).
4
Western Blot Analysis of Myosin and Tubulin
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A total of 10 μg of protein was loaded into each lane for SDS-PAGE, and protein was transferred to Bio-Rad Immun-Blot® PVDF membranes and blocked with 5% milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Primary antibodies against pan-myosin heavy chain (mouse monoclonal A4.1025; EMD Millipore, 05-716) and α-tubulin (rabbit monoclonal EP1332Y; Abcam, ab52866) were diluted 1:1000 in 5% bovine serum albumin in TBST. Secondary antibodies coupled to Alexa Fluor 680 nm fluorophores against mouse IgG and rabbit IgG were diluted 1:10,000 in 5% bovine serum albumin in TBST. Band intensities were analyzed using the gel analysis tool in ImageJ [24 (link)].
5
Western Blot Analysis of Polyglutamate Proteins
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Proteins were separated using a CriterionTM XT precast gel (4–12% Bis-Tris, (Biorad, Hercules, CA, USA)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using the CriterionTM Blotter (Biorad, Hercules, CA, USA). Membranes were incubated with rabbit anti-CCP5 (1:1000, Ab118621, Abcam, Cambridge, MA, USA), mouse anti-polyglutamate (B3, 1:2000, Sigma-Aldrich), mouse anti-glutamate (GT335, 1:4000, Adipogen, San Diego, CA, USA), rabbit anti-long-chain polyglutamate (polyE, 1:4,000, Adipogen), mouse anti-myc (1:1200, Clontech, Mountain View, CA, USA) or rabbit anti-α-tubulin (EP1332Y, 1:3000, Abcam) antibodies. Immunoreactivity of proteins was visualized with Supersignal® West Pico Chemiluminescence Substrate (Thermo, Rockford, IL, USA) following incubation with HRP-labeled sheep anti-mouse (1:2000, GE Healthcare Sciences, Pittsburgh, USA) or donkey anti-rabbit IgG (1:10,000, GE Healthcare Sciences) antisera.
6
Western Blotting of Polyglutamate Proteins
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Proteins were separated using a Criterion XT precast gel (4–12% Bis-Tris, (Biorad, Hercules, CA, USA)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using the Criterion Blotter (Biorad, Hercules, CA, USA). Membranes were incubated with mouse anti-glutamate (GT335, 1:4000, Adipogen), rabbit anti-long-chain polyglutamate (polyE, 1:4,000, Adipogen), or rabbit anti-α-tubulin (EP1332Y, 1:3000, Abcam) antibodies. Immunoreactivity of proteins was visualized with Supersignal West Pico Chemiluminescence Substrate (Thermo, Rockford, IL, USA) following incubation with HRP-labeled sheep anti-mouse (1:2000, GE Healthcare Sciences, Pittsburgh, USA) or donkey anti-rabbit IgG (1:10,000, GE Healthcare Sciences) antisera.
7
Metabolomic Analysis Protocols and Reagents
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Methanol (MeOH), water, formic acid (Optima LC/MS grade) and glutamine were purchased from Thermo-Fisher Scientific (Illkirch, France). Isotope metabolite standards including 17α-Hydroxyprogesterone-d8 (2,2,4,6,6,21,21,21-d8), L-Thyroxine-13C6, Succinic acid-2,2,3,3-d4, Pyruvic acid-1-13C and DL-Alanine-15N with >98% purity were acquired from Sigma Aldrich (St. Quentin Fallavier, France) as well as oligomycin, carbonyl cyanide 4‐(trifluoromethoxy) phenylhydrazone (FCCP), antimycin A and aspartate. All antibodies (ab42364, EP1332Y, ab186695 and ab186696), the NAD/NADH and ATP Assay Kit (ab65348 and ab83355) were obtained from Abcam (Paris, France) and the Mitotracker® green from Molecular Probes (Oregon, USA). Tris-Glycine Gel was purchased from Life Technologies (Illkirch, France) and DMEM-F12 from Jacques Boy Institute of Biotechnology (Reims, France). The DMEM medium supplemented with FBS (fetal bovine serum) was acquired from PAN-biotech (Wimborne, UK) and the Seahorse XFe Base Medium from Agilent Technologies (Santa Clara, CA, USA).
8
Immunofluorescence Staining Methods
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For immunofluorescence experiments, different fixation methods were exploited depending on the antibodies used.
For immunocytochemistry of MTs, cells were extracted for 1 min. in pre-warmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in BRB80 buffer (BRB80 buffer: 80mM Pipes, 1mM EGTA and 4mM MgCl2, pH 6.8) and subsequently fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of cytochrome C, cells were fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of EB1, cells were fixed in ice-cold methanol for 10min. After fixation, cells were washed with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed again with PBS and subsequently blocked for 1hr with 3% BSA in PBS.
Cells were incubated with primary antibody diluted in 3% BSA in PBS for 1hr at RT, washed with PBS and incubated with secondary antibody diluted in 3% BSA in PBS for 1hr at RT. After washing with PBS, cells were dipped in MQ, air-dried and mounted on microscopy slides using Prolong Diamond (Molecular Probes). The following primary antibodies were used in this study: Cytochrome C (6H2.B4, BD Biosciences), EB1 (5/EB1 BD Biosciences), acetylated tubulin (6-11B-1, Sigma-Aldrich), alpha-tubulin (EP1332Y, Abcam), alpha-tubulin (B-5-1-2, Sigma), tyrosinated tubulin (YL1/2, Abcam) and detyrosinated tubulin (AB3210, Merck).
For immunocytochemistry of MTs, cells were extracted for 1 min. in pre-warmed extraction buffer (0.3% Triton X-100 and 0.1% glutaraldehyde in BRB80 buffer (BRB80 buffer: 80mM Pipes, 1mM EGTA and 4mM MgCl2, pH 6.8) and subsequently fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of cytochrome C, cells were fixed in pre-warmed 4% PFA in PBS for 10min. For immunocytochemistry of EB1, cells were fixed in ice-cold methanol for 10min. After fixation, cells were washed with PBS, permeabilized with 0.25% Triton X-100 in PBS, washed again with PBS and subsequently blocked for 1hr with 3% BSA in PBS.
Cells were incubated with primary antibody diluted in 3% BSA in PBS for 1hr at RT, washed with PBS and incubated with secondary antibody diluted in 3% BSA in PBS for 1hr at RT. After washing with PBS, cells were dipped in MQ, air-dried and mounted on microscopy slides using Prolong Diamond (Molecular Probes). The following primary antibodies were used in this study: Cytochrome C (6H2.B4, BD Biosciences), EB1 (5/EB1 BD Biosciences), acetylated tubulin (6-11B-1, Sigma-Aldrich), alpha-tubulin (EP1332Y, Abcam), alpha-tubulin (B-5-1-2, Sigma), tyrosinated tubulin (YL1/2, Abcam) and detyrosinated tubulin (AB3210, Merck).
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