Retina tissue was homogenized in 30 mM of Tris-HCl lysis buffer (pH 7.5) containing 10 mM EGTA, 5 mM EDTA, 1% Triton X-100, 250 mM sucrose, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and protease inhibitor. Protein (150µg) was incubated overnight at 4 °C with 2 µg of the anti-S100A4 antibody and the normal rabbit IgG control antibody (Cat. No. AB-105-C, R&D Systems). Prewashed protein A/G plus agarose beads (20 μl of bead slurry; Santa Cruz Biotechnology, Inc.) were then added to the mixture and rolled for 1 h at 4 °C. After centrifugation, immunoprecipitates were washed 4X with lysis buffer. After the last wash, the resultant pellets were resuspended in 20 μl of 2X Laemmli’s sample buffer and then denatured for 5 min at 95 °C, separated by gel electrophoresis, and transferred to polyvinyl difluoride membranes. This was followed by immunoblotting with an anti-RAGE antibody (1:500; sc-5563; Santa Cruz Biotechnology Inc.) and detection with chemiluminescence plus a Luminol HRP substrate. To evaluate the lane-loading control, the blots were stripped and the S100A4 protein was detected with the anti-S100A4 antibody.
Sc 5563
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-5563 is a laboratory equipment item manufactured by Santa Cruz Biotechnology. It is designed for use in scientific research and analysis. The core function of Sc-5563 is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab 105 c, by R&D Systems (1 mentions)
Ab3611, by Abcam (1 mentions)
Sc 2020, by Santa Cruz Biotechnology (1 mentions)
Af1145, by R&D Systems (1 mentions)
Na9340v, by GE Healthcare (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Apocynin, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Glp 1r, by Santa Cruz Biotechnology (1 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
Sc 23959, by Santa Cruz Biotechnology (1 mentions)
3 protocols using sc 5563
1
Retina Tissue Immunoprecipitation and RAGE Detection
2
Detection of RAGE Protein Expression
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Cells were analyzed for human FL-RAGE expression by WB as already described [35] (link) using a goat polyclonal antibody against the extracellular domain of human RAGE (α-RAGE N-term1, 1 µg/ml; cat. AF1145, R&D Systems, Minneapolis, MN, USA) that recognizes rat RAGE as well. For detection of RAGE in rat lung and R3/1 cells lysates two additional different rabbit polyclonal antibodies against the extracellular (α-RAGE N-term2, 0.4 µg/ml; cat. sc-5563, Santa Cruz Biotechnology, California, USA) and the intracellular (α-RAGE C-term, 1 µg/ml; cat. ab3611, Abcam, Cambridge, UK) domains of human RAGE were used. Both antibodies recognize rat RAGE as well.
The membranes were blocked in TBST (10 mM Tris, pH 7.4; 0.5 mM NaCl; 0.1% Tween 20) containing 5% powdered skimmed milk for 1 hour (h) at rt. The blots were first probed with the indicated primary antibodies diluted in TBST with 5% powdered skimmed milk over night at 4°C, and then with horseradish peroxidase-conjugated anti-rabbit (1∶5000; cat. NA9340V, GE Healthcare) or anti-goat (1∶5000; cat. sc-2020, Santa Cruz Biotechnology) secondary antibodies. Proteins were visualized by an enhanced chemiluminescence (ECL) detection system (cat. RPN2106, GE Healthcare). An antibody agaist GAPDH (0.4 µg/ml; cat. sc-25778, Santa Cruz Biotechnology) was used on the same membranes after stripping and served as loading control.
The membranes were blocked in TBST (10 mM Tris, pH 7.4; 0.5 mM NaCl; 0.1% Tween 20) containing 5% powdered skimmed milk for 1 hour (h) at rt. The blots were first probed with the indicated primary antibodies diluted in TBST with 5% powdered skimmed milk over night at 4°C, and then with horseradish peroxidase-conjugated anti-rabbit (1∶5000; cat. NA9340V, GE Healthcare) or anti-goat (1∶5000; cat. sc-2020, Santa Cruz Biotechnology) secondary antibodies. Proteins were visualized by an enhanced chemiluminescence (ECL) detection system (cat. RPN2106, GE Healthcare). An antibody agaist GAPDH (0.4 µg/ml; cat. sc-25778, Santa Cruz Biotechnology) was used on the same membranes after stripping and served as loading control.
3
Oxidative Stress Assays in Cell Culture
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All reagents for cell culture, GLP-1-(7 (link)–36 (link)) amide, Hoechst 33258 and apocynin (NADPH oxidase inhibitor) were purchased from Sigma (St. Louis, MO, USA). The Annexin V-FITC/propidium iodide (PI) apoptosis detection kit was purchased from Invitrogen (Carlsbad, CA, USA). The cell counting kit-8 (CCK-8), ROS and superoxide anion assay kits were purchased from the Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit anti-p47phox (SC-14015), rabbit anti-p22phox (SC-20781), β-actin (SC-47778), and primary antibodies against RAGE (SC-5563), p53 (SC-126), Bax (SC-23959) and exendin(9 (link)–39 (link)) (SC-364387), the antagonist for receptor of GLP-1 (GLP-1R), were all purchased from Santa Cruz Biotechnology, Inc. (Delaware, CA, USA). The caspase-3 and caspase-9 activity assay kits were obtained from BD Biosciences (Franklin Lakes, NJ, USA).
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