Melan-a melanocytes were lysed with RIPA buffer and 1X protease inhibitor cocktail (PIC) (Sigma-Aldrich). The protein extracts were separated at 200 V using SDS-PAGE and the separated proteins were then transferred to PVDF membranes (Pall, Glen Cove, NY, USA) at 25 V for 2 h. To analyze protein expression, the PVDF membranes were immunoblotted overnight at 4 °C with antibodies to β–actin (Sigma), MyoVa (Cell Signaling Technology, Danvers, MA, USA), Rab27a (Santa Cruz, Dallas, TX, USA), Mlph (Protein Tech Group Inc., Chicago, IL, USA), Tubulin (Cell Signaling Technology) and GR (Cell Signaling Technology). Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Bethyl Laboratories, Montgomery, TX, USA) or anti-mouse antibodies (Bio-Rad, Hercules, CA, USA) were then incubated at room temperature for 1 h. Protein expression was detected using chemiluminescence ECL solution (Thermo, Waltham, MA, USA) and visualized with Chemi-Doc XRS (Bio-Rad).
Anti mouse antibodies
Manufactured by Bio-Rad
Sourced in United States
Anti-mouse antibodies are laboratory reagents used to detect and analyze proteins or other biomolecules in samples derived from mice. These antibodies specifically bind to mouse-derived proteins, enabling their identification and quantification in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Ecl chemiluminescence solution, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Rab27a, by Santa Cruz Biotechnology (1 mentions)
Transfer apparatus, by Bio-Rad (1 mentions)
Ibright cl1000, by Thermo Fisher Scientific (1 mentions)
Mouse anti human β actin, by Santa Cruz Biotechnology (1 mentions)
Ecl system, by Bio-Rad (1 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
Ecl plus western blotting kit, by GE Healthcare (1 mentions)
Pre stained protein marker, by Bio-Rad (1 mentions)
5 protocols using anti mouse antibodies
1
Melanocyte Protein Expression Analysis
2
Western Blot Analysis of ABCB11 in HepG2 Cells
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Total proteins from HepG2 cells were prepared and run on 10% SDS-PAGE and transferred to a PVDF membrane using a transfer apparatus following the standard protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 1 h the membrane was washed once with TBST and incubated overnight at 4°C with rabbit antibodies against human ABCB11 (Affinity, Catalog #DF 9278) 1: 2000 dilution; mouse anti-human β-actin (Santa Cruz Cat.# SC4778), dilution 1:1000. The membrane was washed three times (TBST) and incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit (Santa Cruz Cat# SC-2004)/anti-mouse antibodies (Cat.#SC-2005) for 2 h. Blots were washed with TBST four times and developed with the ECL system (Bio-Rad, US Cat.#170-5060) according to the manufacturer's protocol. The western blot images were acquired using iBright CL1000 (Invitrogen, Thermo Fisher Scientific).
3
In Vitro Ubiquitination Assay Protocol
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All in vitro ubiquitination assays (except those involving discharge assays in Figs 2c and 5 and Supplementary Fig. 3 ) were performed as follows: E1 (0.2 μM) was mixed with UbcH5c (0.5 μM), E3 ligase (0.5–1 μM), ubiquitin (3–5 μM), ATP (3 mM) and 1 μM of histone H2A, H2A/H2B dimer or NCPs. The reactions were incubated at 32 °C for 90 min (unless otherwise stated) in buffer 50 mM Tris/HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 μM ZnCl2 and 1 mM TCEP. Final salt concentration in the assays was normally ~150 mM, unless otherwise stated. Western blot analysis was performed using anti-H2A (Millipore 07–146, 1:1,000 dilution) or anti-ubiquitin (Santa Cruz P4D1, 1:1,000 dilution) antibodies, followed by secondary HRP-conjugated anti-rabbit or anti-mouse antibodies respectively (BioRad, 1:10,000 dilution).
4
Immunoblotting Analysis of Chloroplast Proteins
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For immunoblotting analysis, approximately 10 µg of total proteins and chloroplast proteins were prepared and separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins on the gel were transferred to polyvinylidene fluoride (PVDF) membrane via semi-dry transfer imprinting and incubated in blocking buffer containing 20 mM of trihydrochloric acid (pH 7.5), 500 mM of sodium chloride and 5% skim milk, and then further incubated with 1 RV 5000 diluted polyclonal antibody (Swedish Agrisera, Vännäs, Sweden) at room temperature for 1 h. Anti-rabbit or mouse antibodies (Bio-Rad, Hercules, CA, USA) conjugated with horseradish peroxidase or anti-mouse antibodies (Bio-Rad) conjugated with horseradish peroxidase were used as secondary antibodies. After incubating with the second antibody for 1 h, the signals were detected using an ECL plus Western blotting kit (General Electric Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instruction.
5
Quantifying XTH/XET Protein Levels
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To determine XTH/XET protein levels, 20 μg of cell wall proteins were separated in 12% sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis. Anti-XTH/XET (Agrisera, Vännäs, Sweden) were used as primary antibodies (diluted 1/500) and anti-mouse antibodies (Bio-Rad, Hercules, CA, United States) were used as the secondary antibodies. Immuno-blotting was performed according to the standard protocol. Bands for XTH/XET located at 33 kDa were determined on the basis of a pre-stained protein marker (Bio-Rad) as the reference. The relative protein levels were quantified by densitometry analysis using Image-Lab 5.2. software (Bio-Rad).
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