GC cells were laid in 10 cm culture dishes and cultured to 75% to 90% confluency and harvested in lysis buffer containing proteases and phosphatase inhibitors, leave it at 4 °C for about 45 min. The protein was quantified by BCA analysis. Then the proteins were isolated on SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF) membranes, these membranes were probed using primary antibodies and secondary antibodies according to the supplier's recommendations: DPYS (1:500, Proteintech, 13,237–1-AP, Wuhan, Hubei), NT5E (1:1000, Abcam, ab133582, Suite Cambridge, USA), UPP1 (1:1000, Abcam, ab128854, Suite Cambridge, USA), β-actin (1:1000, #3700, CST, USA), anti-rabbit secondary antibody (Abcam, ab150077, Cambridge, USA) and anti-mouse secondary antibodies (Bio-Rad, 1706516, Hercules, CA). Finally, enhanced chemiluminescence (ECL) was used to observe the results.
Anti mouse secondary antibodies
Manufactured by Bio-Rad
Sourced in United States
Anti-mouse secondary antibodies are laboratory reagents used to detect and visualize target proteins in various immunoassays. These antibodies are designed to specifically bind to the Fc region of mouse primary antibodies, enabling signal amplification and detection of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Mab1501, by Merck Group (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Phosstop phosphate inhibitor cocktail, by Roche (2 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Fortis Life Sciences (2 mentions)
Mouse ant gfp antibody, by Roche (2 mentions)
Bis sulfosuccinimidyl suberate bs3, by Thermo Fisher Scientific (2 mentions)
Complete lysis m, by Roche (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Mini protean tgx precast gel, by Bio-Rad (2 mentions)
Mouse anti gfp antibody, by Roche (2 mentions)
Ab133582, by Abcam (1 mentions)
Ab128854, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Mouse anti vinculin antibody, by Merck Group (1 mentions)
Chemiluminescence reagent for western blotting, by Bio-Rad (1 mentions)
Fulvestrant i e ici182 780, by Bio-Techne (1 mentions)
Dmem with and without phenol red, by Merck Group (1 mentions)
4 protocols using anti mouse secondary antibodies
1
Western Blot Analysis of Proteins
2
Immunoblotting and Immunoprecipitation of RNA Granule Proteins
3
Evaluation of Estrogen Receptor and Ubiquitin in BT-474 and MDA-MB-231 Cells
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BT-474 and MDAMB231 were purchased by ATCC (Manassas, VA, USA). DMEM (with and without phenol red) and fetal calf serum were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bradford protein assay kit, as well as anti-mouse secondary antibodies, were obtained from Bio-Rad (Hercules, CA, USA). Antibodies against ERα (F-10, mouse) and ubiquitin (P4D1, mouse) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-vinculin (mouse) antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemiluminescence reagent for Western blotting was obtained from BioRad Laboratories (Hercules, CA, USA). Fulvestrant (i.e., ICI182,780) was purchased by Tocris (USA). All the other products were from Sigma-Aldrich. Analytical- or reagent-grade products were used without further purification. The identity of the BT-474 and MDAMB231 cell lines was verified by STR analysis (BMR Genomics, Italy).
4
Immunoblotting and Immunoprecipitation of RNA Granule Proteins
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Cells were lysed using cOmplete Lysis-M (Roche) with the addition of PhosSTOP Phosphate Inhibitor Cocktail (Roche) and protein concentration was determined using a colorimetric assay. 50 μg of each sample was ran on 4-20% or 7.5% Mini-Protean® TGX™ precast gels (Bio-Rad) and proteins were transferred onto 0.2-μm PVDF or 0.45-μM nitrocellulose membranes (Bio-Rad). Membranes were immunoblotted with rabbit anti-DDX6 antibody (A300-461A, Bethyl), rabbit anti-DCP2 antibody (A302-597A, Bethyl), mouse anti-GFP antibody (clones 7.1 and 13.1, Roche), rabbit anti-SQSTM1/p62 antibody (A302-857A), mouse anti-LC3 antibody (NB600-1384, Novus) and mouse anti-actin (MAB1501, Millipore). Membranes were then probed with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (Bethyl) or anti-mouse secondary antibodies (Jackson) and proteins were visualized using Clarity™ Western ECL Substrate (Bio-Rad). For immunoprecipitation, 1 mg of total protein from GFP-DCP2-expressing HeLa cells lysate was precipitated using mouse ant-GFP antibody (Roche) attached to Dynabeads® Protein G (Invitrogen) cross-linked by bis(sulfosuccinimidyl) suberate (BS3) (Invitrogen) for 2 h at room temperature. Samples were then washed 3 times, prepared for gel electrophoresis and run on gels as described above. Lysate input was used as a control and immunoblotted accordingly to demonstrate equal loading.
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