MAK034 Coenzyme A Assay Kit 产品说明书

Coenzyme A Assay Kit

Catalog Number MAK034

Storage Temperature –20 C

TECHNICAL BULLETIN

Product Description

Coenzyme A (CoA) is an essential metabolic cofactor

synthesized from cysteine, pantothenate, and ATP.

CoA plays important roles in many metabolic pathways,

including the tricarboxylic acid cycle, and the synthesis

and oxidation of fatty acids. One of the main functions

of CoA is the carrying and transfer of acyl groups.

Acylated derivatives, for example acetyl-CoA, are

critical intermediates in many metabolic reactions. CoA

levels can be altered during starvation, and in

conditions such as cancer, diabetes, and alcoholism.

The Coenzyme A Assay kit is an easy and convenient

assay to measure CoA level in a variety of biological

samples. CoA concentration is determined by an

enzymatic assay, which results in a colorimetric

(570 nm) /fluorometric (

Ex

= 535 nm/

Em

= 587 nm)

product, proportional to the CoA present. Typical

detection range for this kit is 0.2–10 nmoles of CoA.

Components

The kit is sufficient for 100 assays in 96-well plates.

Coenzyme A Assay Buffer 25 mL

Catalog Number MAK034A

Fluorescent Peroxidase Substrate 0.2 mL

Catalog Number MAK034B

Conversion Enzyme Mix 1 vl

Catalog Number MAK034C

Coenzyme A Substrate Mix 1 mL

Catalog Number MAK034D

Acyl Coenzyme A Developer 1 vl

Catalog Number MAK034E

Coenzyme A Standard, 10 mole 1 vl

Catalog Number MAK034F

Reagents and Equipment Required but Not

Provided.

 96 well flat-bottom plate – It is recommended to use

black plates with clear bottoms for fluorescence

assays and clear plates for colorimetric assays.

Cell culture or tissue culture treated plates are not

recommended.

 Fluorescence or spectrophotometric multiwell plate

reader.

 10 kDa Molecular Weight Cut-Off (MWCO) Spin

Filter

Precautions and Disclaimer

For R&D use only. Not for drug, household, or other

uses. Please consult the Safety Data Sheet for

information regarding hazards and safe handling

practices.

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure

water for the preparation of reagents. To maintain

reagent integrity, avoid repeated freeze/thaw cycles.

Coenzyme A Assay Buffer – Allow buffer to come to

room temperature before use.

Fluorescent Peroxidase Substrate – Used as a probe in

both fluorimetric and colorimetric assays. Warm to

room temperature before use. Mix well by pipetting.

Store at –20 C, protected from light and moisture.

For fluorimetric assays, dilute an aliquot of the

Fluorescent Peroxidase Substrate probe 4-fold with

Coenzyme A Assay Buffer just prior to use. This will

reduce the background of the fluorescence assay.

2

Conversion Enzyme Mix and Acyl Coenzyme A

Developer – Reconstitute each in 220 L of

Coenzyme A Assay Buffer. Mix well by pipetting,

then aliquot and store at –20 C. Use within

2 months of reconstitution.

Coenzyme A Standard - Reconstitute in 100 L of

water to generate 100 mM solution. Mix well by

pipetting, then aliquot and store at –20 C. Use

within 2 months of reconstitution.

Storage/Stability

The kit is shipped on wet ice and storage at –20 C,

protected from light, is recommended.

Procedure

Coenzyme A Standards for Colorimetric Detection

Dilute 10 L of the 100 mM Coenzyme A Standard with

990 L of water to prepare a 1 mM (1 nmole/L)

standard solution. Add 0, 2, 4, 6, 8, and 10 L of the

1 mM Coenzyme A standard solution into a 96-well

plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well

standards. Add Coenzyme A Assay Buffer to each well

to bring the volume to 40 L.

Coenzyme A Standards for Fluorometric Detection

Prepare a 1 mM standard solution as for the

colorimetric assay. Dilute 10 L of the 1 mM Coenzyme

A standard solution with 90 L of water to make a

0.1 mM Coenzyme A standard solution. Add 0, 2, 4, 6,

8, and 10 L of the 0.1 mM Coenzyme A standard

solution into a 96-well plate, generating 0 (blank), 0.2,

0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add

Coenzyme A Assay Buffer to each well to bring the

volume to 40 L.

Sample Preparation

Tissue samples (20–40 mg) should be rapidly

homogenized with 100 L of ice-cold PBS or other

buffer (pH 6.5–8). Because enzymes in the sample may

interfere with the sample reading, it is recommended to

deproteinize the samples using a 10 kDa cut-off spin

filter. Add between 1–40 L deproteinized samples into

duplicate wells of a 96-well plate and bring to a final

volume of 40 L with Coenzyme A Assay Buffer.

For unknown samples, it is suggested to test several

sample dilutions to ensure the readings are within the

linear range of the standard curve.

Assay Reaction

1. Add 10 L of the Coenzyme A Substrate Mix

and 2 L of Conversion Enzyme Mix to each

standard and sample well. Mix well.

Note: Long chain acyl-CoAs in the sample can

generate background in the assay. If significant

amounts of acyl-CoAs are in the sample it is

advised to do an Acyl-CoA background control.

Prepare a blank well for each sample by omitting

the Conversion Enzyme from the reaction. The

Acyl-CoA blank background should be subtracted

from CoA readings.

2. Incubate at 37 C for 30 minutes.

3. Set up the Master Reaction Mix according to the

scheme in Table 1 for both the colorimetric and

fluorimetric assays. For fluorimetric assays, use

2 L of the 4-fold diluted probe to decrease the

assay background. For colorimetric assays, use

2 L of undiluted (neat) probe.

50 L of the Master Reaction Mix is required for

each reaction (well).

Table 1.

Master Reaction Mix

Reagent Master Reaction Mix

Coenzyme A Assay Buffer

46 L

Acyl-CoA Developer

2 L

Fluorescent Peroxidase

Substrate probe

2 L

4. Add 50 L of the Master Reaction Mix to each of

the wells. Mix well using a horizontal shaker or by

pipetting and incubate the reaction for 30 minutes

at 37 C. Protect the plate from light during the

incubation.

5. For colorimetric assays, measure the absorbance

at 570 nm (A

570

). For fluorometric assays, measure

fluorescence intensity (

Ex

= 535/

Em

= 587 nm).

Results

Calculations

The background for either assay is the value obtained

for the 0 (blank) Coenzyme A Standard. Correct for the

background by subtracting the blank value from all

readings. Background values can be significant and

must be subtracted from all readings.

Use the values obtained from the appropriate

Coenzyme A standards to plot a standard curve. The

amount of Coenzyme A present in the samples may be

determined from the standard curve.

Note: A new standard curve must be set up each time

the assay is run.

3

Concentration of Coenzyme A

S

a

/S

v

= C

S

a

= Amount of Coenzyme A in unknown sample

(nmole) from standard curve

S

v

= Sample volume (L) added into the wells.

C = Concentration of Coenzyme A in sample

Coenzyme A molecular weight: 767.5 g/mole

Sample Calculation

Amount of Coenzyme A (S

a

) = 2 nmole

Sample volume (S

v

) = 40 L,

Concentration of Coenzyme A in sample

2 nmole/40 L = 0.05 nmole/L

0.05 nmole/L 767.5 ng/nmole= 38.4 ng/l.

4

Troubleshooting Guide

Problem Possible Cause

Assay Buffer Ice Cold

Omission of step in procedure

Plate reader at incorrect wavelength

Assay Not Working

Type of 96 well plate used

Samples prepared in different buffer

Samples were not deproteinized

Samples with erratic

readings

Cell/Tissue culture samples were

incompletely homogenized

Samples used after multiple freeze-thaw

cycles

Presence of interfering substance in the

sample

Use of old or inappropriately stored

samples

Improperly thawed components

Lower/Higher

Readings in Samples

and Standards

Use of improperly stored reagents

Allowing the reagents to sit for extended

times on ice

Incorrect incubation times or temperatures

Incorrect volumes used

Use of partially thawed components

Pipetting errors in preparation of standards

Pipetting errors in the reaction mix

Non-linear Standard

Curve

Air bubbles formed in well

Standard stock is at incorrect

concentration

Calculation errors

Substituting reagents from older kits/lots

Samples measured at incorrect

wavelength

Unanticipated results

Samples contain interfering substances

Sample readings above/below the linear

range

Suggested Solution

Assay Buffer must be at room temperature

Refer and follow Technical Bulletin precisely

Check filter settings of instrument

For fluorescence assays, use black plates

with clear bottoms. For colorimetric assays,

use clear plates

Use the Assay Buffer provided or refer to

Technical Bulletin for instructions

Use a 10 kDa MWCO spin filter to

deproteinize samples

Repeat the sample homogenization,

increasing the length and extent of

homogenization step.

Aliquot and freeze samples if needed to use

multiple times

If possible, dilute sample further

Deproteinize samples

Use fresh samples and store correctly until

use

Thaw all components completely and mix

gently before use

Always check the storage conditions and

store the components appropriately

Always prepare fresh Master Reaction Mix

before use

Refer to Technical Bulletin and verify correct

incubation times and temperatures

Use calibrated pipettes and aliquot correctly

Thaw and resuspend all components before

preparing the reaction mix

Avoid pipetting small volumes

Prepare a Master Reaction Mix whenever

possible

Pipette gently against the wall of the tubes

Always refer to the dilutions in the Technical

Bulletin

Recheck calculations after referring to

Technical Bulletin

Use fresh components from the same kit

Check the equipment and filter settings

If possible, dilute sample further

Deproteinize samples

Concentrate or dilute samples so readings

are in the linear range

SS,LS,MF,MAM 01/21-1

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