Cells were pelleted and washed three times by PBS. Fractionation of subcellular proteins were collected using Subcellar Protein Fractionation Kit (Thermo Scientific, 78840). Cell lysates were prepared in lysis buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 0.2% n-dodeyl-D-Maltoside, 1 mM N-Ethylmaleimide, 1% Triton-100, 0.02 mM MG132, and Pierce protease inhibitors (Thermo Scientific, A32965). Protein samples were loaded on 4–20% SDS-PAGE gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The following primary antibodies were used for protein blotting: mouse anti-c-Myc antibody (Sigma-Aldrich, 05–724MG), mouse anti-Flag antibody (Sigma-Aldrich, F3165), Rabbit anti-ITFG1 antibody (ITFG1_Ab1) (Invitrogen, PA5–54067), mouse anti-ITFG1 antibody (ITFG1_Ab2) (R&D Systems, MAB89001), Rabbit anti-RUVBL1 antibody (Proteintech, 10210–2-AP), Rabbit anti-GAPDH antibody (Cell Signaling Technology, 2118). Signals were developed with HRP-labeled secondary antibodies (Bio-Rad). Blots were developed using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using ChemiDoc MP Imaging System (Bio-Rad).
Hrp labeled secondary antibodies
Manufactured by Bio-Rad
Sourced in United States
HRP-labeled secondary antibodies are a type of laboratory reagent used to detect and visualize target proteins in various immunoassays. These antibodies are conjugated with the enzyme horseradish peroxidase (HRP), which catalyzes a colorimetric or chemiluminescent reaction, allowing for the detection and quantification of the target proteins.
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Lab products found in correlation
Mouse anti flag antibody, by Merck Group (1 mentions)
Pierce protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Mouse anti c myc antibody, by Merck Group (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Rabbit anti gapdh antibody, by Cell Signaling Technology (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using hrp labeled secondary antibodies
1
Subcellular Protein Fractionation and Western Blot Analysis
2
Protein Expression Analysis by Western Blot
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The whole-cell proteins were extracted using RIPA lysis buffer (Beyotime, China). SDS-PAGE (10%) gel (EpiZyme, China) was used to separate the proteins, and proteins were transferred to PVDF membranes. We probed GPR87, E-cadherin, N-cadherin, Vimentin, and GAPDH with the corresponding antibodies at 4 °C for 12 h and detected them with chemiluminescence 2 h after incubation with HRP-labeled secondary antibodies (Bio-Rad, USA). Antibodies are listed in Table S3 .
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