Proteins from CD2+-T cells and CD14+ monocytes of MM patients and controls were solubilized with lysis buffer [50 mM Tris(Tris(hydroxymethyl)aminomethane)-HCl (pH 8.0), 150 mM HCl, 5 mM ethylenediaminetetraacetic acid, 1% NP40 and 1 mM phenylmethyl sulfonyl fluoride]. The protein concentration was measured by DC™ Protein Assay (Bio-Rad, California, U.S.). Cell proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and afterwards transferred to nitrocellulose membranes (Millipore, Massachusetts, U.S.). The membranes were incubated overnight at 4°C with mouse anti-LIGHT, mouse anti RANKL (Abcam, Cambridge, U.K.) and rabbit anti-total ERK (Santa Cruz Biotechnology, Texas, U.S.). After incubation with appropriate IRDye 800 cw goat anti-mouse and IRDye 800 cw goat anti-rabbit secondary Ab (LI-COR, Nebraska, U.S.), the membranes were detected on an Odissey scanner (LI-COR, Nebraska, U.S.).
Mouse anti rankl
Manufactured by Abcam
Sourced in Italy
Mouse anti-RANKL is a monoclonal antibody that specifically binds to the receptor activator of nuclear factor kappa-B ligand (RANKL) in mice. RANKL is a key regulator of bone resorption and is involved in the differentiation and activation of osteoclasts.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (1 mentions)
Odissey scanner, by LI COR (1 mentions)
Rabbit anti total erk, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Mouse anti p62, by Solarbio (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Mouse anti rank, by Abcam (1 mentions)
Supersignal west femto maximum sensitivity substrate reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Rabbit anti traf6, by Abcam (1 mentions)
Liteablot turbo luminol reagents, by Euroclone (1 mentions)
Hyperfilm ecl film, by Euroclone (1 mentions)
Rabbit anti osterix, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti tgf β, by Abcam (1 mentions)
Rabbit anti cxcl12, by Abcam (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Cell Signaling Technology (1 mentions)
3 protocols using mouse anti rankl
1
Western Blot Analysis of LIGHT and RANKL
2
Quantifying Osteoclastogenesis Signaling Proteins
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Fresh GCTB tissues were harvested using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). The total protein of each specimen (30 mg/lane) were separated by SDS-PAGE (10% gels), and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore). Β-actin was used as a loading control. The membrane was blocked with 5% bovine serum albumin (BSA, Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 40 min, and subsequently incubated with mouse anti-p62 (1:1,500; cat. no. ab56416; Abcam), mouse anti-RANKL (1:1,000; cat. no. ab45039; Abcam), mouse anti-RANK (1:1,000; cat. no. ab13918; Abcam) and mouse anti-β-actin (1:5,000; cat. no. ab6276; Abcam) primary antibodies overnight at 4°C. The membranes were then incubated with peroxidase-conjugated goat anti-mouse IgG (1:20,000; cat. no. A4416; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. Protein bands were visualized using SuperSignal™ West Femto Maximum Sensitivity Substrate reagents (Thermo Fisher Scientific, Inc.). The relative gray value of the immune reactive bands was compared using ImageJ software (version 1.46, National Institutes of Health).
3
Western Blot Analysis of Bone Cells
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Proteins from total BMCs and from BMSCs were extracted in cell lysis buffer (Cell Signaling, EuroClone) after 2 days of culture, and the concentration was determined by the BCA protein assay reagent (Pierce, EuroClone). Western blotting was performed as previously described (Sabbieti et al. 2010) (link). Membranes were immunoblotted in blocking buffer with specific antibodies: rabbit anti-TNFα and rabbit anti-NF-κB (BioLegend, Microtech SrL, Napoli, Italy) both diluted 1:500; mouse anti-RANKL, rabbit anti-TRAF6, rabbit anti-CXCL12 and rabbit anti-TGFβ (Abcam, Prodotti Gianni) all diluted 1:600; rabbit anti-PPRγ (Santa Cruz Biotechnology, Inc. DBA) diluted 1:300 and rabbit anti-Osterix (Santa Cruz Biotechnology, DBA) diluted 1:300. After washing with PBS-T, blots were incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG or with HRP-conjugated rabbit anti-mouse IgG (Cell Signaling, EuroClone) both diluted 1:50,000. Immunoreactive bands were visualized using LiteAblot Turbo luminol reagents (EuroClone) and Hyperfilm-ECL film (EuroClone) according to the manufacturer's instructions. To normalize the bands, filters were stripped and re-probed with a monoclonal anti-α-tubulin (Sigma-Aldrich). Band density was quantified densitometrically.
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