Protein samples were loaded on polyacrylamide gel at the concentration of 10 μg/well and separated by SDS/PAGE, using 4–20% precast gel (Mini-PROTEAN® Precast Gels, Bio-Rad, Hercules, CA, USA). Proteins were transferred on nitrocellulose membranes and immunoblotted by monoclonal antibodies anti-s100A8, anti-s100A9 and ANXA2 (R&D systems). Protein-bands were detected by chemiluminescent Pierce™ ECL Western blotting substrate (Bio-Rad) and analyzed by ChemiDoc™ XRS (Bio-Rad). Densitometric analysis was conducted using ImageJ software.
Anti s100a8
Manufactured by R&D Systems
Sourced in United States
Anti-S100A8 is a laboratory reagent that can be used to detect and measure the presence of the S100A8 protein. S100A8 is a calcium-binding protein that plays a role in various cellular processes. The Anti-S100A8 reagent can be used in research applications that involve the study of S100A8 and its functions.
Automatically generated - may contain errors
Lab products found in correlation
Anti s100a9, by R&D Systems (3 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Mini protean precast gel, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Bio-Rad (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Merck Group (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Xct slc7a11, by Cell Signaling Technology (1 mentions)
Lat1 slc7a5, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase hrp coupled anti rabbit 7074 and anti mouse 7076, by Cell Signaling Technology (1 mentions)
Antipuromycin antibody clone 12d10, by Merck Group (1 mentions)
Odyssey fc, by LI COR (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
4 protocols using anti s100a8
1
SDS-PAGE and Western Blot Analysis
2
Immunoblotting of S100A8 and S100A9
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Healthy and tumor parts of fresh frozen samples were homogenized in RIPA buffer containing proteinase inhibitors, followed by protein concentration measurement using Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories Inc.). 50 μg protein was loaded onto reducing 15% SDS gel, followed by transfer onto PVDF membrane. Membranes were blocked with 5% nonfat dry milk in PBS-0.05% Tween-20 for overnight at 4° C. Immunoblotting was performed with primary antibodies anti-S100A8 and anti-S100A9 (R&D Systems) diluted in blocking solution incubating the membrane for 1 h at room temperature, followed by incubation with HRP conjugated anti-mouse secondary antibody (Sigma) for 1 h at room temperature. Signal was detected by Bio-Rad Clarity western ECL (Bio-Rad Laboratories Inc.) and autoradiography.
3
Quantitative Western Blotting of S100A8 and S100A9
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Western blotting was performed as previously described [34 (link)]. Briefly, lysates from dorsal skin and gut tissues were lysed in RIPA with protease inhibitor and quantified using the BCA protein assay (Thermo Scientific, Waltham, MA, USA, 23225). Then, 20 μg of protein were loaded into an SDS-PAGE gel (10% or 12%) and transferred to PVDF membranes (Millipore, Burlington, MA, USA, IPVH00010). The nonspecific binding of proteins was blocked with 5% skim milk for 1 h at room temperature, then the membranes were incubated with the primary antibodies anti-S100A8 (R&D system, Minneapolis, MN, USA, af2059) and anti-S100A9 (R&D system, af2065) overnight at 4 °C. Their corresponding secondary antibodies were incubated with the membranes for 1 h at room temperature. The membranes were scanned with a ChemiDoc imaging system (Bio-Rad Laboratories).
4
Tasquinimod and Venetoclax Combination in AML
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KG-1a and MOLM-13 cells were cultured at a density of 2.5 × 105 cells/mL and were treated with tasquinimod for 48 h. For the combination experiment, AML cells were treated with tasquinimod for 24 h, followed by venetoclax treatment for additional 24 h. Cells were lysed in cell lysis buffer including protease (Roche) and phosphatase inhibitors (Sigma). Western blot analysis on these cell lysates was performed as previously described [21 (link)]. The following primary antibodies were used: p-mTOR (#5536), mTOR (#2983), p-P70S6K (#9204), P70S6K (#2708), p-S6K (#4868), S6K (#2217), p-4E-BP1 (#2855), 4E-BP1 (#9644), p-eIF2a (#9721), eIF2a (#9722), ATF4 (#11815), p21 (#2974), p27 (#3688), S100A9 (#72590), xCT/SLC7A11 (#12691), LAT1/SLC7A5 (#32683), BCL-2 (#2870), c-MYC (#5605) and horseradish peroxidase (HRP)-coupled anti-rabbit (#7074) and anti-mouse (#7076) secondary antibodies; all purchased from Cell Signaling Technology (Boston, MA). GRP78 (#sc-13968) was from Santa Cruz Biotechnology, anti-S100A8 (#MAB4570) from R&D, and anti-puromycin antibody, clone 12D10 (#618582) was from Sigma-Aldrich. The bands were visualized and captured using Pierce ECL Western Blot Substrate (Thermo Scientific) and LI-COR Odyssey Fc (Bad Homburg, Germany). Pixel densities were quantified using Image J.
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