Mouse Adiponectin ELISA

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Pre-warm all reagents to room temperature prior to setting up the assay.	-Do not let wells dry before proceeding to the next step. If an automated machine is used for the assay, use a gentle wash program for all washing steps. -For best result all additions should be completed within one hour. -Remove any air bubble formed in the well after acidification of substrate solution because bubbles interfere with spectrophotometric readings.

Upstream tips
-Pre-warm all reagents to room temperature prior to setting up the assay.
Protocol tips
-Do not let wells dry before proceeding to the next step. If an automated machine is used for the assay, use a gentle wash program for all washing steps. -For best result all additions should be completed within one hour. -Remove any air bubble formed in the well after acidification of substrate solution because bubbles interfere with spectrophotometric readings.

Publication protocol

Serum was collected by cardiac puncture 10 weeks following primary tumor regression, and selected biomarkers were evaluated by enzyme-linked immunosorbent assay (ELISA) according to manufacturers’ protocols. All samples were run in duplicate. The adipokine multiplex panel (insulin, leptin, resistin, tissue plasminogen activator 1) was assessed using the MADKMAG-71K assay kit (EMD Millipore, Billerica, MA, USA). Adiponectin levels were assessed using the EZMADP-60K assay kit (EMD Millipore). IGF-1 and insulin-like growth factor-binding proteins 1–7 (IGFBP1–7) were assessed using the 22-IG1MS-E01 assay kit (ALPCO, Salem, NH, USA) and the MIGFBPMAG-43K multiplex kit (EMD Millipore), respectively. Estradiol, testosterone, and sex hormone-binding globulin were measured using the ES180S-100 (Calbiotech, El Cajon, CA, USA), 55-TESMS-E01 (ALPCO), and CSB-E08233m (Biotrend, Destin, FL, USA) assays, respectively. Hepatocyte growth factor (HGF) was measured using the MHG00 assay kit (R&D Systems, Minneapolis, MN, USA). The inflammatory markers C-reactive protein (CRP), monocyte chemoattractant protein (MCP)-1, and corticosterone were assessed using the RH971CRP01MR (BioVendor, Brno, Czech Republic), MJE00 (R&D Systems), and 55-CORMS-E01 (ALPCO) assay kits, respectively. Multiplex ELISA plates were read on a Luminex 100 plate reader (Luminex Corp., Austin, TX, USA) and analyzed using Exponent software (Texture Technologies, Hamilton, MA, USA). Single ELISAs were read on the Molecular Devices M2 plate reader and analyzed using SoftMax Pro software (Molecular Devices, Sunnyvale, CA, USA).

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Manufacturer protocol

Download the product protocol from Merck Millipore for Mouse Adiponectin ELISA below.

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