{"id":493,"date":"2018-07-30T17:22:59","date_gmt":"2018-07-30T21:22:59","guid":{"rendered":"https:\/\/www.lifetein.com\/blog\/?p=493"},"modified":"2024-11-16T21:59:34","modified_gmt":"2024-11-17T02:59:34","slug":"how-to-do-competition-binding-assay","status":"publish","type":"post","link":"https:\/\/www.lifetein.com\/blog\/how-to-do-competition-binding-assay\/","title":{"rendered":"How to do competition binding assay?"},"content":{"rendered":"\n
\"ELISA
ELISA for peptide antigen antibody reactions<\/figcaption><\/figure>\n\n\n

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as co-receptors to activate FGF receptor (FGFR) signaling. <\/span><\/p>\n

A series of peptides were synthesized by LifeTein and used for the competition binding assay. Both the KL1 and KL2 domains of \u03b2-Klotho participate in ligand interaction. The FGF19 peptide was used for alanine scanning mutagenesis. It was found that a single amino acid mutation in either region was sufficient to abolish \u03b2-Klotho binding. FGF19 and FGF21 function through \u03b2-Klotho to regulate glucose and lipid metabolism. <\/span><\/p>\n

How to perform the solid-phase binding assay<\/b><\/span><\/p>\n

1. The 96-well plates were coated overnight at 4 \u00b0C with 2 \u00b5g\/mL of antibody in PBS. <\/span><\/p>\n

2. Plates were washed twice with PBST and blocked with 3% (w\/v) BSA in PBS for 1.5 hours at room temperature. <\/span><\/p>\n

3. The conditioned media containing \u03b2-Klotho were added to the plates and incubated for 1.5 hours at room temperature. <\/span><\/p>\n

4. Plates were washed a few times. <\/span><\/p>\n

5. The peptide mutation FGF21 and an anti-\u03b2-Klotho antibody were biotinylated with EZ-Link Sulfo-NHS-LC-Biotin at the indicated concentrations. <\/span><\/p>\n

6. After washing, streptavidin-HRP was used for detection. <\/span><\/p>\n

7. EC<\/span>50<\/span>\u00a0values were determined.<\/span><\/p>\n

How to do a competition binding assay?<\/b><\/span><\/p>\n

1. The WT and mutant peptides were custom synthesized and purified (>95% purity) by LifeTein. <\/span><\/p>\n

2. Binding of FGF19 and FGF21 peptides to \u03b2-Klotho was assessed. <\/span><\/p>\n

3. The \u03b2-Klotho ECD 6 \u00d7 His, varying amounts of FGF19 and 21 peptides, and biotinylated human FGF19 or FGF21 protein were prepared. <\/span><\/p>\n

4. The streptavidin donor beads and nickel chelate acceptor beads were added to the plates. <\/span><\/p>\n

5. Plates were incubated for 3 hours at room temperature, protected from light, and read on the Plate Reader.<\/p>\n

Reference: <\/span>Scientific Reports, <\/i>volume<\/b><\/span> <\/b>8<\/b>, Article\u00a0number:\u00a011045\u00a0(2018)<\/span><\/p>\n

\"receptor-binding-assay\"

receptor-binding-assay<\/p><\/div>","protected":false},"excerpt":{"rendered":"

Endocrine fibroblast growth factors (FGFs) require Klotho transmembrane proteins as co-receptors to activate FGF receptor (FGFR) signaling. A series of peptides were synthesized by LifeTein and used for the competition binding assay. 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