Coenzyme A Detection

实验概要

The experiment provided  an  easy, convenient assay to measure the CoA level in a variety of  biological samples. In the assay, free CoA is specifically utilized to  generate products which react with OxiRed Probe to generate color  (Lambda = 570 nm) and fluorescence (Ex=535/Em=587 nm). The assay can  detect 0.1 to 10 nmol of CoA (2.5-250 µM concentration range) in a  variety of samples.

实验原理

Coenzyme  A (CoA) is composed of units derived from cysteine, pantothenic acid,  and ATP. It plays important roles in the synthesis and oxidation of  fatty acids, pyruvate oxidation in the citric acid cycle and many other  biological processes. One of the main functions of Coenzyme A is the  carrying and transfer of acyl groups. One of the most important acyl  groups transferred is the acetate group, in which case the molecule is  called acetyl-Coenzyme A. The acetyl group eventually finds itself  incorporated into a variety of molecules such as cholesterol,  acetylcholine, melatonin, heme and the TCA cycle intermediates.

主要试剂

Acyl CoA Developer Green

CoA Assay Buffer

CoA Standard Yellow

CoA Substrate Purple

Conversion Enzyme Mix Blue

DMSO (anhydrous) Brown

OxiRed Probe Red

实验步骤

1.    Reagent Preparation:

1)   OxiRed Probe: Dissolve with 220 µl of DMSO (provided, need to warm up >18°C to become liquid) before use. Mix well.

2)   Conversion Enzyme Mix, Acyl CoA Developer: Dissolve with 220 µl CoA Assay Buffer. Pipette up and down to completely dissolve.

3)   CoA Standard: Dissolve in 100 µl dH2O to generate 100 mM (100 nmol/µl) CoA Standard solution.

2.    CoA Assay Protocol:

1)   CoA Standard Curve Preparations:

Colorimetric assay:

Dilute  the CoA Standard to 1 nmol/µl by adding 10 µl of the Standard to 990 µl  of dH2O, mix well. Add 0, 2, 4, 6, 8, 10 µl into a series of standards  wells on a 96 well plate. Adjust volume to 40 µl/well with CoA Assay  Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the CoA Standard.

Fluorometric  assay: Dilute the CoA Standard to 1 nmol/µl as for the colorimetric  assay. Then dilute another 10-fold to 0.1 nmol/µl by taking 10 µl into  90 µl of dH2O. Mix well. Add 0, 2, 4, 6, 8, 10 µl into a series of  standards wells on a 96 well plate. Adjust volume to 40 µl/well with CoA  Assay Buffer to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well CoA  standard.

2)   Sample  Preparation: Tissue samples (20-40 mg) should be rapidly homogenized  with 100 µl ice cold PBS or other buffer (pH 6.5-8). Enzymes in samples  may interfere with the assay. We suggest deproteinizing your sample  using a perchloric acid/KOH protocol or 10 kd molecular weight cut off  spin columns. Add 1-40 µl sample into 96-well plate, bring volume to 40  µl with Assay Buffer. We suggest testing several doses of your samples  to ensure the readings are within the standard curve range.

3)   CoA Conversion: Add 10 µl of Substrate, 2 µl of Conversion Enzyme Mix* to each standard and sample. Mix well.

*Long  chain acyl-CoA’s in the sample can generate background in the assay. If  a significant amount of acyl-CoA is in your sample, do a background  control; omit Conversion Enzyme from the reaction. The acyl-CoA  background should be subtracted from CoA readings.

4)   Incubate for 30 minutes at 37°C.

5)   Develop:  Mix enough reagent for the number of samples and standards to be  performed: For each well, prepare a total 50 µl Reaction Mix containing:

46 µl CoA Assay Buffer

2 µl Acyl-CoA Developer**

2 µl OxiRed Probe***

Add 50 µl of the Reaction Mix to each well containing the CoA Standard and test samples.

**The Acyl-CoA developer recognizes C8 or longer fatty acid chain to generate signal.

***Use  0.5 µl OxiProbe in the fluorometric Assay to decrease fluorescence  background and thus increase detection sensitivity, significantly.

6)    Incubate for 30 minutes at 37°C, protect from light.

7)   Measure OD at 570 nm for the colorimetric assay, or Ex/Em=535/589 for the fluorometric assay.

8)   Calculation:  Correct background by subtracting the value of the 0 CoA control from  all sample readings (Note: The background reading can be significant and  must be subtracted from sample readings). Plot the standard curve. Then  apply the sample readings to the standard curve to get CoA amount in  the sample wells.

The CoA concentrations in the test samples:

C = Ay/Sv (nmol/µl; or µmol/ml; or mM)

Where:

Ay is the amount of CoA (nmol) in your sample from the standard curve.

Sv is the sample volume (µl) added to the sample well.

CoA molecular weight: 767.5g/mol.

注意事项

       General Considerations:

Protect Kit components from light.

Warm CoA Assay Buffer to room temperature before use.

Briefly centrifuge all small vials prior to opening.

Store reconstituted OxiRed Probe at -20°C, protect from light and moisture.

Store reconstituted Conversion Enzyme Mix and Acyl CoA Developer at -20°C. Use within two months.

Keep reconstituted CoA Standard cold while in use and then store at -20°C.