Creatine Assay Kit, BioAssay™

Creatine is present in vertebrates and helps to supply energy to muscle. In humans and animals, approximately half of creatine originates from food (mainly from fresh meat). Creatine supplementation has been investigated as a possible therapeutic approach for the treatment of muscular, neuromuscular, neurological and neurodegenerative diseases.|Simple, direct and automation-ready procedures for measuring creatine are popular in research and drug discovery. Creatine assay is based on enzymatic reactions leading to formation of a pink colored product. The optical density at 570 nm or fluorescence intensity at lem/ex=590/530 nm is directly proportional to the creatine concentration in the sample.||Key Features:|High sensitivity and wide linear range. Use 10ul sample. Linear detection range 4 to 1000uM (colorimetric) or 0.5 to 50uM (fluorometric).|Homogeneous and simple procedure. Simple “mix-and-measure” procedure allows reliable quantitation of creatine within 30 minutes.||Applications:|Direct Assays: creatine in biological samples (e.g. serum, plasma, urine, saliva etc).||Kit Contents: (100 tests in 96-well plates)|Assay Buffer: 20ml Enzyme A: 120ul|Enzyme B: 220ul Standard: 400ul 20mM creatine|Dye Reagent: 220ul|Storage conditions. The kit is shipped chilled. Store all components at -20°C. Shelf life: 9 months after receipt. Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|Sample preparation: SH-group containing reagents (e.g. mercaptoethanol, DTT) and EDTA may interfere with this assay and should be avoided in sample preparation. Solid samples can be extracted by homogenization in distilled water (dH2O) and filtered or centrifuged. Liquid samples (e.g. serum, plasma and urine) can be assayed directly.||Colorimetric Procedure:|1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge tubes before opening. Prepare a 1000uM creatine Standard Premix by mixing 15ul of the 20mM Standard and 285ul dH2O. Dilute Standard as follows.|No Premix+dH2O Vol (ul) Creatine (uM)|1 100ul+0ul 100 1000|2 60ul+40ul 100 600|3 30ul+70ul 100 300|4 0ul+100ul 100 0|Transfer 10ul standards into separate wells of a clear, flat-bottom 96-well plate.|Transfer 10ul of each sample into two separate wells, one serving as a sample blank well (RBLANK) and one as a sample well (RSAMPLE).|2. Enzyme Reaction. For each standard and sample well, prepare Working Reagent by mixing 90ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B and 1ul Dye Reagent. Add 90ul Working Reagent to the four Standards and the Sample Wells. Prepare blank control reagent by mixing 90ul Assay Buffer, 1ul Enzyme B and 1ul Dye Reagent (i.e. no Enzyme A). Add 90ul Blank control reagent only to the Sample Blank Wells. Tap plate to mix. Incubate 30 min at room temperature.|3. Read OD570nm.||Fluorometric Procedure:|The Fluorometric Procedure: is the same as for the Colorimetric Assay:, except that (1) the detection range is up to 50uM creatine and (2) a black, flat-bottom 96-well plate is used. Creatine standards of 0, 15, 30 and 50uM are prepared. After incubation for 30 min at room temperature, read fluorescence intensity at lex=530 nm and lem=590 nm.||Calculation:|Subtract the standard values from the blank value (#4) and plot the DOD or DF against standard concentrations. Determine the slope and calculate the creatine concentration of Sample,|[Creatine] =|RSAMPLE–RBLANK|Slope (uM-1)|× n (uM)|RSAMPLE and RBLANK are optical density or fluorescence intensity readings of the Sample and Sample Blank, respectively. n is the sample dilution factor.|Note: if the calculated creatine concentration is higher than 1000uM in the Colorimetric Assay: or 50uM in the Fluorometric Assay:, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n.|Conversions: 1000uM creatine equals 13.1 mg/dL or 131 ppm.||Materials Required, But Not Provided:|Pipeting devices, and clear flat-bottom 96-well plates and optical density plate reader for Colorimetric Assay:s; black flat-bottom 96-well plate and fluorescence intensity plate reader for Fluorometric Assays.