Product Name:	Lactose Assay Kit (BA0126)
SKU:	BA0126
Detection Method:	Colorimetric (570 nm) or fluorimetric (λex/em = 530/585 nm)
Detection Range:	17 to 2000 µM (colorimetric); 6 to 100 µM (fluorimetric)
Sample Type:	Milk and other biological samples; food and beverage products
Species Reactivity:	All
Assay Time:	30 minutes
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	Store all components at -20°C.
Shelf Life:	6 months after receipt.
Shipping:	Gel Pack

Lactose is cleaved by lactase and coupled enzyme reactions convert the resulting galactose into a coloured product. The colour intensity at 570 nm, or fluorescence intensity at λex/em = 530/585 nm, is directly proportional to the lactose concentration.

Uses as little as 20 µL of sample.
Linear detection range in a 96-well plate of 17 to 2000 µM lactose (colorimetric) and 6 to 100 µM (fluorimetric).
Colorimetric and fluorimetric read-out options for flexibility and sensitivity.

Assay of lactose in milk and other biological samples.
Drug discovery and pharmacology: effects of drugs on lactose metabolism.
Food and beverages: lactose in food and beverage products.

Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1	Notes: glycerol and thiol-containing reagents (e.g. β-mercaptoethanol, dithiothreitol) interfere and should be avoided; a sample blank is required for samples containing galactose; addition of Working Reagent should be quick and mixing brief but thorough, and a multi-channel pipettor is recommended.
2	Sample treatment: clear milk by mixing 600 µL milk with 100 µL 6 N HCl, centrifuge 5 minutes at 14,000 rpm, transfer 300 µL supernatant to a clean tube and neutralise with 50 µL 6 N NaOH (dilution factor n = 1.36).
3	Equilibrate all components to room temperature and reconstitute the Lactase and Enzyme Mix each with 120 µL distilled water; keep the reconstituted reagents refrigerated or on ice during the experiment.
4	Prepare 400 µL of 2000 µM standard by mixing 40 µL of the 20 mM standard with 360 µL distilled water and dilute in water as described in the standard table.
5	Transfer 20 µL of each standard and 20 µL of each sample into separate wells of a clear flat-bottom 96-well plate, adding a sample blank well for samples known to contain galactose.
6	For each reaction well, mix 85 µL Assay Buffer, 1 µL Lactase, 1 µL Enzyme Mix and 1 µL Dye Reagent (for sample blanks prepare a control Working Reagent without the Lactase); transfer 80 µL Working Reagent into each well, tap to mix and incubate 30 minutes at room temperature.
7	Read the optical density at 570 nm (550-585 nm).
8	For fluorimetric assays, prepare a 100 µM standard (5 µL of 20 mM standard plus 995 µL water), dilute to 100, 80, 60, 40, 30, 20, 10 and 0 µM, use a black 96-well plate, add 80 µL Working Reagent, incubate 30 minutes and read fluorescence at λex = 530 nm and λem = 585 nm.

Subtract the blank value (water, standard #8) from the standard values and plot the change in signal against concentration to determine the slope. Colorimetry: [Lactose] = (OD_SAMPLE - OD_BLANK) / Slope x n (µM). Fluorimetry: [Lactose] = (RFU_SAMPLE - RFU_BLANK) / Slope x n (µM). The blank is water when no galactose is present or the sample blank when galactose is present, and n is the dilution factor. If the calculated concentration exceeds 2000 µM (colorimetric) or 100 µM (fluorimetric), dilute in water and repeat. Conversions: 1 mM lactose equals 34.2 mg/dL, 0.0342% or 342 ppm.