Creatine Assay Kit

Catalog No. ABIN1000304

Product Details

Target: Creatine

Detection Range: 4-1000 μM

Minimum Detection Limit: 4 μM

Application: Biochemical Assay (BCA)

Sample Type: Serum, Plasma, Urine, Saliva

Characteristics: High sensitivity and wide linear range. Use 10 µL sample. Linear detection range 4 to 1000 µM (colorimetric) or 0.5 to 50 µM (fluorimetric).
Homogeneous and simple procedure.
Simple mix-and-measure procedure allows reliable quantitation of creatine within 30 minutes.

Components: Assay Buffer: 20 mL. Enzyme A: 120 µL. Enzyme B: 220 µL. Standard: 400 µL 20 mM creatine. Dye Reagent: 220 µL.

Material not included: Pipeting devices, and clear flat-bottom 96-well plates and optical density plate reader for colorimetric assays, black flat-bottom 96-well plate and fluorescence intensity plate reader for fluorimetric assays.

Application Details

Application Notes: Direct Assays: creatine in biological samples (e.g. serum, plasma, urine, saliva etc).

Protocol: 1. Standards and Samples. Equilibrate all components to room temperature. Briefly centrifuge tubes before opening. Prepare a 1000 µMcreatine Standard Premix by mixing 15 µL of the 20 mM Standard and 285 µL dH2O. Transfer 10 µL standards into separate wells of a clear, flat-bottom 96- well plate. Transfer 10 µL 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 90 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B and 1 µL Dye Reagent. Add 90 µL Working Reagent to the four Standards and the Sample Wells. Prepare blank control reagent by mixing 90 µL Assay Buffer, 1 µL Enzyme B and 1 µL Dye Reagent (i.e. no Enzyme A). Add 90 µL Blank control reagent only to the Sample Blank Wells. Tap plate to mix. Incubate 30 min at room temperature.
3. Read OD570nm. Fluorimetric Procedure The fluorimetric procedure is the same as for the colorimetric assay, except that (1) the detection range is up to 50 µMcreatine and (2) a black, flat-bottom 96-well plate is used. Creatine standards of 0, 15, 30 and 50 µMare prepared. After incubation for 30 min at room temperature, read fluorescence intensity at ex = 530 nm and em = 590 nm.

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.

Calculation of Results:

Subtract the standard values from the blank value (#4) and plot the OD or F against standard concentrations. Note: if the calculated creatine concentration is higher than 1000 µM in the colorimetric assay or 50 µM in the fluorimetric assay, dilute sample in dH2O and repeat assay. Multiply result by the dilution factor n.
Conversions: 1000 µM creatine equals 13.1 mg/dL or 131 ppm.

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Schoborg, Hodgman, Anderson, Jewett: "Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis." in: Biotechnology journal, Vol. 9, Issue 5, pp. 630-40, (2014) (PubMed).

Target Details

Target: Creatine

Target Type: Amino Acid

Background: Quantitative determination of creatine by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 30 min.

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. This 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 gamma em/ex = 590/530 nm is directly proportional to the creatine concentration in the sample.