For the RIP assay, the cell lysates were homogenized according to the protocol specified by the Magna RIP™RNA-Binding Protein Immunoprecipitation Kit (MILLIPORE Catalog No. 17-701). MCF-7 and MDA-MB-231 cells were lysed in RIP lysis buffer containing proteinase inhibitors cocktail tablets (Roche, Cat. 04693116001) and RNAse inhibitor (MILLIPORE, Cat. CS203219) and incubated with anti-IGF2BP3- (Merck, Cat. 03-198), or anti-Ago2 (GeneTex, Cat. GTX60370) or IgG-coupled protein beads for 6 h or overnight at 4 °C. After stringently washing the beads with washing buffer, the bound RNA was purified and subjected to RT–PCR assays.
Proteinase inhibitors cocktail tablets
Manufactured by Roche
Sourced in United States
Proteinase inhibitors cocktail tablets are a lab equipment product designed to inhibit the activity of various proteases, enzymes that break down proteins. These tablets are typically used in research and experimental settings to preserve the integrity of protein samples during analysis and processing.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Cell Signaling Technology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Minute cytoplasmic and nuclear extraction kit, by Thermo Fisher Scientific (2 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Rnase inhibitor, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
4 protocols using proteinase inhibitors cocktail tablets
1
RIP Assay for RNA-Binding Proteins
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells or tissue using RIPA buffer (Cell Signaling, USA) containing proteinase inhibitors cocktail tablets (Roche,Catalog # 04693116001) and nuclear protein was isolated using MinuteTM Cytoplasmic and Nuclear Extraction Kit (ThermoFisher, USA). For a typical WB analysis, 10–20 μg of protein lysates were separated using 6–12% SDS-PAGE, and transferred to PVDF membranes (EMD Millipore, USA). The membranes were incubated with the selected primary antibodies and then secondary antibodies. Signals were detected by an ECL kit (Pierce, USA) according to the manufacturer’s instructions.
3
Protein Extraction and Western Blot Analysis
4
Western Blot and Immunohistochemistry Protocol
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For WB analysis, total protein from cells or tissue was extracted using RIPA buffer (Cell Signaling, USA) supplemented with proteinase inhibitors cocktail tablets (Roche, Catalog # 04693116001). WB analysis was carried out according to the standard protocol. Briefly, 10–20 μg of protein lysates were first separated using 6–12% SDS-PAGE. Afterward, the protein was transferred to PVDF membranes (EMD Millipore, USA), which were then incubated with the selected primary antibodies and then secondary antibodies. Expression of candidate proteins were detected by an ECL kit (Pierce, USA). For immunohistochemistry analysis, sections of tissue or cells were prepared, fixed, and stained using primary antibodies. After extensive wash, the tissue or cells were incubated with the Fluor or HRP labeled secondary antibodies, and then colorized with the avidin-biotin-peroxidase complex (ABC) method following the manufacturer’s protocol. Images were acquired using a laser scanning confocal microscope FV1000 (Olympus, Japan).
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