Creatine Assay Kit

Catalog #K635-100; 100 assays; Store Kit at –20^oC)

I. Introduction:

Creatine is an endogenous compound whose function is to maintain a high ATP/ADP ratio, by way of its phosphorylated form and creatine kinase. Creatine supplementation has been used in the treatment of muscular, neurological and neurodegenerative diseases, as well as a sport performance enhancer. Detection of creatine level has wide applications in research and development. BioVision’s Creatine Assay Kit provides an accurate, convenient measure of creatine in a variety of biological samples. In the assay, creatine is enzymatically converted to sarcosine which is then specifically oxidized to generate a product that converts a colorless probe to an intensely red color (_max = 570 nm), and highly fluorescent (Ex/Em = 538/587 nm) product. Creatine is therefore easily detected by either colorimetric or fluorometric methods. Detection range 0.001 – 10 mM Creatine.  

II. Kit Contents:

III. Reconstitution of Reagents:

1. Creatine Assay Buffer: Ready to use as supplied. It may be stored at 4°C or -20^oC.

1. 
   

   Creatine Probe: Dissolve in 220 µl DMSO (provided). Vortex to dissolve. Store at -20°C, protect from light and moisture. Stable for at least 2 months.

   
   


2. 
   

   Creatinase, Creatine Enzyme mix: Reconstitute with 220 µl of Assay Buffer. Keep on ice during use. Store at -20°C when not in use. Aliquot each and store until needed. Freeze/thaw should be limited to one time.

   
   


3. 
   

   Creatine Standard: Reconstitute with 100 l of dH₂O to generate 100 nmol/l Creatine Standard. Dissolve completely. Store at -20°C, stable for 2 months.

   
   

IV. Assay Protocol:

1. 
   

   Prepare Standard: Mix 10 µl reconstituted creatine standard with 990 l of Assay Buffer, mix to generate 1 nmol/l standard working solution. Add 0, 2, 4, 6, 8, 10 µl of the working solution to 6 consecutive wells. Bring the volume to 50 µl each well with Assay Buffer.

   
   

If a more sensitive method is desired, fluorescence assay can be utilized. Further dilute the standard 10-100 fold, and follow the same procedure as for the colorimetric assay.

2. 
   

   Prepare Samples: High concentrations of proteins may interfere with the assay. Samples containing proteins may be filtered through a 10k MW cut-off filter (BioVision Cat.# 10kc-20) prior to assay. Add 0-50 µl of sample to the wells and bring the volume to 50 µl with Assay buffer.

   
   

Note: For unknown samples, we suggest testing several different dilutions to ensure the readings are in the linear range of the standard curve.

3. 
   

   Prepare Reaction Mix: Prepare enough reaction mix for the standard and samples. For each assay:

   
   

l Assay Buffer

l Creatinase*

l enzyme

l probe

4. Mix well. Add 50 µl of the appropriate Reaction Mix to each standard and sample well, mix. Incubate at 37°C for 1 hr.

*Note: Sarcosine gives background for the assay. For samples which may contain a significant amount of sarcosine, do a background control. Prepare a reaction without the creatinase (replacing the creatinase with 2 l assay buffer).

5. Read the plate in a plate reader at 570 nm, or fluorescence at Ex/Em = 538/587 nm.

V. Calculations:

1. 
   

   Plot Standard Curve: Subtract reagent background from all readings. Plot readings vs. nmoles creatine.

   
   


2. 
   

   Determine sample Creatine concentrations: Subtract the background reading from the creatine assay sample. Apply the creatine reading to the standard curve. Creatine concentration:

   
   

C = Sa/Sv nmol/l, or mM

Where Sa is the sample amount of unknown in nmol from your standard curve.

Sv is the sample volume added to the well in micro-litter.

Creatine Molecular Weight: 131.13.