Total proteins were extracted from cells using Whole Cell Lysis Assay (KeyGEN BioTECH, China) on ice and quantified by bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The western blot was performed according to standard procedures. The following primary antibodies were applied: MIF (Novus Biologicals, USA), TIMP3, PI3K, CD81, TSG101, ALIX (Abcam, USA), caspase-3, caspase-9, P-c-Raf, AKT, P-Akt (Ser473), P-Akt (Thr308), β-actin (Cell Signaling Technology, USA), BAX (Bioss ANTIBODIES, USA), P21 (Proteintech, USA), CDK2 (ImmunoWay, USA), and Cyclin H (ZenBio Science, China). The antibody information is listed in Table S2 . After primary antibody incubation, the membranes were incubated with HRP-labeled goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology, USA). The bands were captured by chemiluminescence (Biosciences, Foster City, CA, USA) through ECL detection system. Finally, the protein expression was analyzed by the software of ImageJ.
Hrp labeled goat anti rabbit or goat anti mouse secondary antibodies
Manufactured by Cell Signaling Technology
Sourced in United States
HRP-labeled goat-anti-rabbit or goat-anti-mouse secondary antibodies are lab equipment used in immunoassays. They serve as detection reagents, binding to primary antibodies and producing a measurable signal through an enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 3, by Cell Signaling Technology (2 mentions)
Caspase 9, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (2 mentions)
P c raf, by Cell Signaling Technology (1 mentions)
P akt thr308, by Cell Signaling Technology (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
Timp3, by Abcam (1 mentions)
Bcl2 associated x (bax), by Bioss Antibodies (1 mentions)
Tsg101, by Abcam (1 mentions)
Whole cell lysis assay, by Keygen Biotech (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Imagequant las 500 chemiluminescence, by GE Healthcare (1 mentions)
Keygen total protein extraction kit, by Keygen Biotech (1 mentions)
Wnt5a, by Cell Signaling Technology (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Tcf1 tcf7, by Cell Signaling Technology (1 mentions)
Alkbh5, by Merck Group (1 mentions)
Cylind1, by Cell Signaling Technology (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
3 protocols using hrp labeled goat anti rabbit or goat anti mouse secondary antibodies
1
Comprehensive Protein Analysis Protocol
2
Quantitative Protein Expression Analysis
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The proteins were extracted from cells using the Keygen total protein extraction kit (Keygen, Biotech, Jiangsu, China) and then quantified by a bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA, USA) as previously described [40 , 41 (link)]. The western blot experiments were performed according to standard procedures. The primary antibodies applied are shown in Supplementary Table 3 . The membranes were incubated with HRP-labeled goat-anti-rabbit or goat-anti-mouse secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). The proteins were detected and visualized using ImageQuant Las500 chemiluminescence (GE, Boston, USA). The protein expression was analyzed by the Image J software (National Institution of Health, USA), and β-actin was used as a control.
3
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from specimens and cells using radioimmunoprecipitation assay lysis buffer with protease inhibitor on ice and quantified by bicinchoninic acid protein assay kit (Thermo, USA). The western blot was performed according to standard procedures. The following specific antibodies were applied: ALKBH5 (Millipore Corporation, USA), SOX2, Wnt5a, β-catenin, C-myc, CylinD1, LEF1, TCF1/TCF7, Met/pro-Met, Caspase-3, Caspase-7, Caspase-8, Caspase-9, MDR1, BCRP1, MRP1, and β-actin (Cell Signaling Technology, USA). The antibody information is listed in Supplementary Table S4 . The membranes were incubated with HRP-labeled goat-anti-rabbit or goat-anti-mouse secondary antibodies (Cell Signaling Technology, USA). The proteins were detected and visualized by chemiluminescence (Biosciences, Foster City, CA, USA). The protein expression was analyzed by the Image J software, and β -actin was used as loading control.
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