N6-isopentenyladenosine | Cytokinin ELISA quantitation kit

Background: This ELISA assay utilise the principle of competitive binding to measure the concentration of hormone in plant extracts. The N6-isopentenyladenosine specific antibodies are precoated to the surface of the reaction wells. The plant extract sample, containing an unknown amount of hormone, is mixed in the reaction well with a known amount of a tracer to react with a limited number of antibodies in the reaction wells. During incubation the hormone in the sample competes with the tracer for the antibody binding sites. Unbound hormone, tracer and plant extract are washed out of the reaction wells. Following substrate addition which reacts with a tracer bound to the antibody and produces a yellow-coloured product. The absorbance of the sample is converted to concentration of hormone by means of a standard curve which is produced by simultaneously treating standards along with the samples.

Reaction wells: Antibody coated and blocked, 5pcs for 480 assays, 60 strips with 8 wells

Tracer: 20-50 µl

Tracer diluent: 5x 250 mM TBS Tris, 10 mM NaCl, 1 mM MgCl2, pH 7.5 stock + 0.02 % NaN₃

Reaction and wash solution: 10x TBS stock+0.02 % NaN₃

Stopping reagent: 2x 5 N KOH stock

 

Important note: Do NOT methylate cytokinin samples as this will significantly decrease the sensitivity of the assay.

Substrate diluent: 10x 500 mM NaHCO₃ stock, pH 9.6+0 0.02 %. 0 0.02 % NaN₃

Substrate: 100 mg

Standards: 600 µl of each: 15.6 pmol, 7.8 pmol, 3.9 pmol, 1.95 pmol, 975 fmol, 488 fmol, 244 fmol, 122 fmol, 61 fmol, 30.5 fmol, 31.2 pmol

Assay development time: 4-5 hours

Sensitivity: 0.01 to 10 pmol/50 µl

Plant extract volume: 50 µl


Note: The antibody has been prepared for riboside. But it binds also free base isopentenyladenine 6-(y,y-Dimethylallylamino)purine. 50% cross-reactivity.