{"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. Both the KL1 and KL2 domains of \u03b2-Klotho … Continue reading →<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":263,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_crdt_document":"","_monsterinsights_skip_tracking":false,"_monsterinsights_sitenote_active":false,"_monsterinsights_sitenote_note":"","_monsterinsights_sitenote_category":0,"footnotes":""},"categories":[4],"tags":[67,131],"class_list":["post-493","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-peptide_synthesis","tag-peptide-receptor","tag-receptor-binding-assay"],"aioseo_notices":[],"_links":{"self":[{"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/posts\/493","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/comments?post=493"}],"version-history":[{"count":0,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/posts\/493\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/media\/263"}],"wp:attachment":[{"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/media?parent=493"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/categories?post=493"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.lifetein.com\/blog\/wp-json\/wp\/v2\/tags?post=493"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}