Fatty Acid Oxidation (FAO) Assay Kit

(No reviews yet) Write a Review
SKU:
BR00001
€599

Description

Fatty Acid Oxidation Kit Overview

The Assay Genie Fatty Acid Oxidation Assay kit is a highly sensitive colorimetric assay for the quantitative measurment of Fatty Acid Oxidation in cells and tissues. This unique assay utilizes a highly soluble Octanoyl-CoA substrate which enables the accurate measurement of Fatty Acid Oxidation versus longer chain less-soluble substrates such as the 18C unsaturated fatty acid Oleate.

Kit Description

  • Fatty acids provide more ATP per carbon than carbohydrates such as glucose. In tissues with high energy requirements such as heart tissue, up to 50 – 70% of energy comes from fatty acid beta-oxidation, which converts fatty acids to acetyl-CoA with concomitant production of FADH2 and NADH in the mitochondria.
  • This strictly aerobic oxidative pathway consists of four distinct steps: acyl-CoA dehydrogenation by acyl-CoA dehydrogenase, hydration by enoyl-CoA hydratase, dehydrogenation by 3-hydroxyacyl-CoA dehydrogenase, and thiolytic cleavage by beta-ketothiolase. The importance of the beta-oxidation system in humans is exemplified by the existence of a group of genetic diseases categorized as fatty acid oxidation disorders (FAODs), which are a heterogeneous group of defects in fatty acid transport and oxidation, and are inherited as autosomal recessive disorders, having a wide range of clinical presentations. In addition, evidence indicates that regulation of activity of FAO during fetal development is not only important for fetal life but also has implications for health and disease in adulthood. The measurement of FAO activity thus provides valuable insights into the pathophysiologic and metabolic basis of many diseases.
  • The Assay Genie non-radioactive FAO assay is based on oxidation of the substrate octanoyl-CoA. Generation of NADH is coupled to the reduction of the tetrazolium salt INT to formazan. The intensity of the red-colored formazan is proportional to increased FAO activity. The assay solution and substrate should be stored in aliquots at -80ºC.
fao-assay-kit-assay-genie.png

Figure: Fatty Acid Oxidation Assay: Tissue lysates were prepared from healthy Hamster hearts and the hearts of the TO2 Hamster model which is associated with heart failure. Lysates were used in the assay and the levels of fatty acid oxidation quantified.

Preparation of cell/tissue extracts:

Step Procedure

1.

Wash ~106 cells twice with ice-cold phosphate-buffered saline (PBS), and remove PBS completely from the cell pellet. Cell pellet should be stored at -80ºC. Tissue sample should be washed with PBS thoroughly to remove blood cells, which can cause inconsistent assay result.

2.

Prepare enough 1x Cell Lysis Solution by diluting 10x Cell Lysis Solution with ice-cold dH2O. Add 50 – 100 µl ice-cold 1x Cell Lysis Solution to cell pellet. Extract cells by pipetting up and down (gently but thoroughly). Leave lysate on ice for 5 min with intermittent gentle agitation. If lysate is viscous, add more 1x Cell Lysis Solution and repeat pipetting. Centrifuge lysate in a refrigerated microfuge for 5 min at maximal speed. Recover supernatant for assay. Tissue is homogenized in 1x Cell Lysis Solution, and lysate is clarified by centrifugation. Use ~25 mg of tissue per 0.5 ml of 1x Cell Lysis Solution for tissue homogenization.

3.

Perform protein assay to determine sample protein concentration. Normalize sample protein concentration by diluting with ice-cold 1x Cell Lysis Solution to 1 – 3 mg/ml. Keep protein sample on ice at all times. Freeze thawed crude protein lysate can exhibit reduced enzyme activity. Lysate should be stored at -80ºC. Note: Do not use a buffer containing reducing agents or SDS.

Reagent thawing:

 Keep thawed FAO Assay Solution and 20x FAO Substrate on ice. Gently agitate solution prior to pipetting. It is important to minimize the time the reagents are thawed. Freeze solutions immediately after use.  

Preparation of control solution and reaction solution:

  • Control solution is prepared by mixing 1 part of dH2O and 20 parts of FAO Assay Solution, e.g. 25 µl dH2O mixed with 500 µl FAO Assay Solution. Keep freshly prepared control solution on ice during assay.
  • Reaction solution is prepared by mixing 1 part of 20x FAO substrate and 20 parts of FAO Assay Solution, e.g. 25 µl 20x FAO Substrate mixed with 500 µl FAO Assay Solution. Keep solution on ice and use immediately. Each protein sample is treated with 50 µl control solution and 50 µl reaction solution in two separate sets.

FAO assay protocol:

Step Procedure

1.

Add 10 µl of each protein sample to a 96-well plate in duplicate. Note: For drug discovery applications, add 1 µl of a drug inhibitor to both wells, and mix with sample by pipetting up and down. 

2.

After all samples have been pipetted to the plate, swiftly add 50 µl control solution to one set of wells and 50 µl reaction solution to another set of wells. Mix contents by gentle agitation for 30 sec. Cover plate and incubate in a humidified 37°C incubator for 1 − 2 hr. FAO activity generates cherry red color in wells. Do not use a CO2 incubator. Note: Increase sample protein concentration and/or incubation time if little red color is detected. 

3.

Stop assay by adding 50 µl 3% Acetic acid (not included in the kit) to each control solution well and reaction  solution well followed by brief gentle agitation. Measure O.D.492 nm using a plate reader.

4.

Subtract control well reading from reaction well reading for each sample. The subtracted O.D. reading is proportional to fatty acid oxidation activity of the sample.

Additional Information

  •  A 3% Acetic acid solution needs to be prepared for reaction termination. The assay solution contains DMSO and iodonitrotetrazolium violet. Please refer to the product page of our website or contact us for MSDS information.  
View AllClose