Oocytes were lysed in 10 µl of 0.1% w/v PBS/PVP with 4 μl Laemmli buffer (Bio-Rad, 161-0747) with β-mercaptoethanol (Gibco 21985023), a phosphatase inhibitor, and protease inhibitor. The oocyte lysates were boiled for 5 min at 95 °C and left on ice, then separated on 10% v/v or 12% v/v polyacrylamide gels and transferred to a Polyvinylidene difluoride (PVDF) membrane (Millipore ISEQ00010). Membranes were blocked in 5% w/v milk for 1 h at room temperature and incubated with primary antibody overnight at 4 °C. The primary antibody and dilutions used were as follows: CPEB1 (Abcam, ab73287, 1:1000), α-tubulin (Sigma-Aldrich, T6074, 1:10,000), T320-pp1 (Abcam, ab62334, 1:30,000). Membranes were washed in TBS-Tween 20 (0.05% v/v) and incubated with HRP-conjugated secondary antibodies, 1:10,000 anti-rabbit IgG (GE Healthcare, NA934V) and 1:10,000 anti-mouse IgG (GE Healthcare NA931V), for 2 h at room temperature. Signals were detected using Clarity Western ECL substrate (Bio-Rad, 170-5061). The images are analyzed with the ImageJ 1.53a.
T6074
Manufactured by Abcam
T6074 is a lab equipment product. It is a glass container designed for use in various laboratory applications. The core function of T6074 is to provide a controlled environment for storage, mixing, or testing of samples or solutions.
Automatically generated - may contain errors
Lab products found in correlation
Ab62334, by Abcam (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Ab23905, by Abcam (1 mentions)
Las af software version 1.6.0 build 1016, by Leica (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Tcs sp5, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Ab109027, by Abcam (1 mentions)
Na934v, by GE Healthcare (1 mentions)
Na931v, by GE Healthcare (1 mentions)
3 protocols using t6074
1
Western Blot Analysis of Oocyte Proteins
2
Immunostaining of Cell Lines
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A549, H1650, H460, Calu-6, PC9 or AALE cells were plated onto poly-D -lysine-coated eight-well glass chamber slides (5,000 cells per well) for immunostaining. The cells were fixed with 10% buffered formalin and indirect immunofluorescence was performed as described56 (link). Primary antibodies used were phospho-TBK1 (Cell signaling #5483S) or CEP170 mouse monoclonal-72-413-1 (Invitrogen #41-3200) at 1:100 dilution or 1:500 dilution of rabbit monoclonal NuMA (EP3976 Abcam #109262), Alpha tubulin (1:2,000, Sigma #T6074), Dynein intermediate chain (Abcam#ab23905) and Kif2b (Abcam #ab98214 or Novus #NBP86002), γ Tubulin (Santa Cruz #sc51715) were used at 1:100 dilution. Anti-rabbit Alexa Fluor-488 or anti- mouse Alexa Fluor-594 (Molecular Probes) was used as secondary antibody. DAPI (Vector Labs) was used to stain the nuclei. Cells were visualized with a DM16000 inverted Leica TCS SP5 tandem scanning confocal microscope with a × 63/1.40NA oil immersion objective. Images and Z-stacks were produced with three cooled photomultiplier detectors and analysed with the LAS AF software version 1.6.0 build 1016 (Leica Microsystems, Germany).
3
Western Blot Analysis of Meiotic Proteins
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Oocytes at indicated times of incubation under maturing conditions were collected in 0.1% w/v PBS/PVP and mixed with Laemmli sample buffer supplemented with β-mercaptoethanol, proteinase inhibitor and phosphatase inhibitor. After boiling at 95°C for 5 min, the lysates were separated on 8% v/v or 12% v/v polyacrylamide gels and transferred to a Polyvinylidene difluoride (PVDF) membrane (Millipore ISEQ00010). Membranes were incubated in 5% w/v milk buffer for 1 h at room temperature and incubated in primary antibody overnight at 4°C. The antibodies used were as follows: CPEB1 (Abcam, ab73287, 1:1000), Cyclin B1 (Cell-Signaling, 4138, 1:1000), Aurora kinase (Cell-Signaling, 2914T, 1:500), MAPK (Cell-Signaling, 4377, 1:1000), α-tubulin (Sigma-Aldrich, T6074, 1:10,000), DDB1(Abcam, ab109027, 1:4000), and T320-PP1 (Abcam, ab62334, 1:30,000). Membranes were washed in 1 x TBST and incubated in the appropriate secondary antibodies (anti-Rabbit, GE Healthcare, NA934V; anti-mouse, GE Healthcare, NA931V) for 2 h at room temperature. Clarity Western ECL substrate (Bio-Rad, 170-5061) was used to develop the membranes, and ImageJ 1.53a was used for quantification for the signals.
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