Total proteins from cell lines and tissues were extracted in a lysis buffer (CAT.78510) (Thermo Fisher Scientific, Rockford, IL) and quantified using Bradford method (CAT.23226) (Thermo Fisher Scientific). Sixty micrograms of protein were separated by SDS-PAGE (10%). After transferring by Trans-Blo ® TurboTM (Bio-Rad, USA), the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies Mig-6 (1: 1000, CAT.11630-1-AP) (protein tech), anti-P62/SQSTM1 (1: 1000,CAT.55274-1-AP)(protein tech), GFP (1:1000,CAT.ab38689) (Abcam), β-actin (1:2000, CAT.20536-1-AP) (protein tech), anti-phospho-JNK (Thr183/Tyr185) (1:1000, CAT.4668) (Cell Signaling Technology, Danvers, MA), anti-LC3b (1:2000, CAT.NB100-2220) (Novus Biologicals USA), anti-JNK (1:1000, CAT.AJ518) (Beyotime Biotechnology). After incubation with peroxidase-coupled anti-mouse or rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 hours, bound proteins were visualized using ECL (Thermo Fisher Scientific) and detected using ChemidocTM MP Imaging Systerm (Bio-Rad, USA). The relative protein levels were calculated based on β-actin as the loading control.
Mig 6
Manufactured by Proteintech
Sourced in United States
The Mig-6 is a laboratory equipment that serves as a high-performance electrophoresis system. It is designed for the separation and analysis of proteins, nucleic acids, and other macromolecules.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3b, by Novus Biologicals (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
β actin, by Proteintech (2 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Thermo Fisher Scientific (1 mentions)
Anti jnk, by Beyotime (1 mentions)
Anti phospho jnk thr183 tyr185, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Anti p62 sqstm1, by Proteintech (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Phospho prb, by Cell Signaling Technology (1 mentions)
Pan s6, by Cell Signaling Technology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Enhanced chemiluminescence ecl detection system, by Bio-Rad (1 mentions)
Anti tgf β2, by Abcam (1 mentions)
Anti p62, by Proteintech (1 mentions)
Sds page, by Bio-Rad (1 mentions)
3 protocols using mig 6
1
Protein Quantification and Western Blot Analysis
2
Protein Extraction and Immunoblotting Assay
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For protein extraction, the assays were performed with 10-cm cell culture plates, and the cells were seeded at 500,000 cells per dish. Briefly, the cells were lysed using NETN buffer [8 (link)] supplemented with phosphatase inhibitors [8 (link)] and followed by three cycles of freezing (in liquid nitrogen) and thawing (in a 37 °C water bath). The protein extracts were separated by SDS-PAGE. The primary antibodies used for immunodetection were for MIG6 (Proteintech, 11630-1-AP), panAKT (Cell Signaling, 4685S, lot 6), AR (Biogenex, 256M), β-Actin (Abcam, ab6276, GR3324554-1), p-AKT (S473) (Cell Signaling, 4058S, lot 14), pRb (Abcam, ab6075, lot 821737), phospho-pRb (Cell Signaling, 9308, lot 13), panS6 (Cell Signaling, 2217S, lot 10), and p-S6 (S235/236) (Cell Signaling, 2211S, lot 23). Horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling, 7076S, lot 32) or anti-rabbit IgG (Cell Signaling, 7074S, lot 28) were used as secondary antibodies. The detection was performed by ImageQuantTM LAS 4000 (GE Healthcare Bio-Sciences AB, Chicago, Illinois, U.S.). Quantification of bands was performed via the LabImage D1 program.
3
Western Blot Analysis of Cellular Proteins
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Total proteins from cell lines were extracted in a lysis buffer (CAT.78510) (Thermo Fisher Scienti c, Rockford, IL) and quanti ed using Bradford method (CAT.23226) (Thermo Fisher Scienti c). Sixty micrograms of protein were separatedby 12% SDS-PAGE (Bio-Rad, USA). After transferring by Trans-Blo ® Turbo™ (Bio-Rad, USA), the polyvinylidene uoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4 °C with the following antibodies Mig-6 (1: 1000, CAT.11630-1-AP) (protein tech), anti-P62 (1:1000,CAT.55274-1-AP) (protein tech), anti-LC3b (1:2000, CAT.NB100-2220) (Novus Biologicals USA),anti-TGF-β2 (1:1000,CAT.ab36495) (Abcam), β-actin (1:1000, CAT.20536-1-AP) (protein tech). After incubation with peroxidase-coupled anti-mouse or rabbit IgG (Zhongshan jinqiao,China) at 37 °C for 2 hours. An enhanced chemiluminescence (ECL) detection system (Bio-Rad, USA) was used to visualize signals following standard protocols. β-actin served as an endogenous protein control for normalization.
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