N6-benzyladenosine | 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-benzyladenosine 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

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, 15.2 fmol

Assay development time: 4-5 hours

Sensitivity: 0.01 to 10 pmol/50 µl

Plant extract volume: 50 µl

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

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Sample clean-up:
Processing of plant extracts required prior ELISA analysis may vary from plant to plant and will depend upon the actual research objectivce. In most cases pigments and lipophilic material needs to be removed by C18 reversed phase chromatography and subsequently by combined DEAE-cellulose-reversed phase C18 columns.

Samples from algae and bacteria can be analyzed directly or after pre-purification.