MG63 and U2OS cells were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 6.8, 32 mM 2-ME, 2% w/v SDS, 10% glycerol) along with protease inhibitor. Protein concentration was determined and 20 µg protein was separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequent to blocking at 37°C with 5% non-fat milk in Tris-buffered saline (TBS) for 30 min, the membranes were incubated with primary antibodies directed against IGF-1R (1:1,000 dilution; cat. no., ab39398) and GADPH (1:1,000 dilution; cat. no., ab8245; both Abcam, Cambridge, UK) overnight at 4°C. Subsequently, the membranes were washed with TBS-Tween 20, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:1,000 dilution; cat. no., ab6785 and ab150077; Abcam) for 1 h at room temperature. An enhanced chemiluminescent system (EMD Millipore) was used to visualize signal bands, and analyzed with Image Lab software (version 3.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Enhanced chemiluminescent system
Manufactured by Merck Group
Sourced in United States, United Kingdom
The Enhanced chemiluminescent system is a laboratory equipment product designed to detect and quantify proteins in Western blot applications. It utilizes chemiluminescent reagents to produce a light signal proportional to the amount of target protein, which can be captured and analyzed using specialized imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab6785, by Abcam (1 mentions)
Bicinchoninic acid protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Myd88, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Bax 50599 2 ig, by Proteintech (1 mentions)
2 protocols using enhanced chemiluminescent system
1
Western Blot Analysis of IGF-1R in MG63 and U2OS Cells
2
Protein Isolation and Western Blot Analysis
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All collected cells were washed twice with precooled PBS, lysed in a radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Shanghai, China), then treated with ultrasound, and centrifuged at 10,000 g at 4°C for 20 min. The protein amount was quantified using a bicinchoninic acid protein assay reagent (Thermo Fisher Scientific, MA, USA). The total protein was isolated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to a polyvinylidene difluoride film (Millipore, Watford, UK). The membrane was immunoblotted overnight at 4°C with anti-toll-like receptor 4 (TLR4) (1 : 300; Abcam, MA, USA), anti-myeloid differentiation primary response 88 (MyD88) (1 : 500; Abcam, MA, USA), NF-KB (p50) (66535-1-IG, Proteintech), BAX (50599-2-IG, Proteintech), Bcl-2 (26593-1-AP, Proteintech), CASPASE3 (19677-1-AP, Proteintech), CASPASE9 (10380-1-AP, Proteintech) and anti-GAPDH (Santa Cruz Biotechnology, CA, USA). After washing in 1 × TBST three times, the horseradish peroxidase-labeled secondary antibody (Jackson ImmunoResearch, PA, USA) was used for detection at room temperature for 1 h. The protein bands were measured using an enhanced chemiluminescent system (Merck Millipore, Watford, UK). Jackson ImmunoResearch.
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