Human HGF Activator, HGFAC ELISA Kit

Human HGFAC ELISA Kit

Keepstreptavidin-HRP at 4Cand protectit from prolonged exposure to light. Aliquot all other reagents and store at-20C to -70C in a manual defrostfreezer.

TheELISA kit is a solid phase sandwich ELISA (Enzyme-Linked ImmunosorbentAssay). It utilizes a monoclonal antibody specific for HGFAC coated on a96-well plate. Standards and samples are added to the wells, and any HGFACpresent binds to the immobilized antibody. The wells are washed and abiotinylated rabbit anti-HGFAC monoclonal antibody is then added, producingan antibody-antigen-antibody “sandwich”. To produce color in proportion tothe amount of HGFAC pre-sent in the sample streptavidin-HRP and TMB substratesolution are loaded. The absorbances of the microwell are read at 450nm.

HGFAC

3083

NP_001519

Q04756

PlatePreparation 1.Dilute the capture antibody to the working concentration in CBS. Immediatelycoat a 96-well microplate with 100ul per well of the diluted captureantibody. Seal the plate and incubate overnight at 4C. 2. Aspirate eachwell and wash with at least 300ul wash buffer, repeating the processtwo times for a total of three washes. Complete removal of liquid at eachstep is essential for good performance. After the last wash, remove anyremaining wash buffer by inverting the plate and blotting it against cleanpaper towels. 3. Block plates byadding 300ul ofblocking buffer to each well. Incubate at room temperature for a minimum of1hour. 4. Repeat theaspiration/wash as in step2. The plates are now ready for sample addition. AssayProcedure 1.Add 100 ul ofsample or standards in sample dilution buffer per well. Seal the plate andincubate 2 hours at room temperature. 2.Repeat the aspiration/wash as in step 2 of plate preparation. 3.Add 100 ul of thedetection antibody, diluted in antibody dilution buffer, to each well. Sealthe plate and incubate 1 hour at room temperature. 4.Repeat the aspiration/wash as in step 2 of plate preparation. 5.Add 100ul ofStreptavidin-HRP to each well. Incubate for 1 hour at room temperature. 6.Repeat the aspiration/wash as in step 2 of plate preparation. 7.Add 200 ul ofsubstrate solution to each well. Incubate for 20 minutes at room temperature (ifsubstrate solution is not as requested, the incubation time should beoptimized). Avoid placing the plate in direct light. 8.Add 50ul of stopsolution to each well. Gently tap the plate to ensure thorough mixing. 9.Determine the optical density of each well immediately, using a microplatereader set to 450nm.