AffiASSAY® ATP Colorimetric & Fluorometric Assay Kit

SKU: AFG-BV-0083

AffiASSAY® ATP Colorimetric & Fluorometric Assay Kit

AffiGEN
$420.70 USD
$420.70 USD
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AffiASSAY® ATP Assay Kit

ATP Colorimetric & Fluorometric Assay Kit

A compact enzymatic assay for ATP quantification in biological samples, with flexible readout by absorbance at OD 570 nm or fluorescence at Ex/Em 535/587 nm.

Catalog No. AFG-BV-0083
100 wells
Colorimetric or fluorometric
Reduced-volume protocol
Research use only

Overview

ATP is the main cellular energy molecule and is widely used as an indicator of metabolic activity, mitochondrial function, cell viability, and tissue energy status. This assay generates a signal proportional to the ATP amount present in the sample and can be measured using either absorbance or fluorescence detection.

Detection modes: OD 570 nm for colorimetric measurement, or Ex/Em 535/587 nm for fluorometric measurement.

Assay Principle

ATP in the sample drives an enzymatic conversion reaction. The resulting intermediate is further processed to generate hydrogen peroxide, which oxidizes a signal probe to produce a colored and fluorescent product. The final signal intensity is proportional to ATP concentration.

ATP assay principle showing ATP conversion and signal detection
Assay principle: ATP-dependent enzymatic conversion followed by signal probe oxidation.

Kit Components

Component names are presented using simplified functional descriptions for a compact product page format.

Component Functional Name Size Storage
Reagent A Assay Buffer 25 mL -20°C or 4°C
Reagent B Signal Probe 0.2 mL -20°C, protect from light
Reagent C ATP Conversion Enzyme Lyophilized -20°C
Reagent D Signal Development Enzyme Mix Lyophilized -20°C
Reagent E ATP Calibrator Lyophilized -20°C

Storage and Handling

  • Store the kit at -20°C.
  • Protect light-sensitive reagents from direct light.
  • Briefly centrifuge vials before opening.
  • Bring liquid reagents to room temperature before use.
  • Keep prepared samples and standards on ice when possible.
  • Avoid repeated freeze-thaw cycles of enzyme reagents.

Reagent Preparation

Reagent C — ATP Conversion Enzyme

Reconstitute with 220 µL Assay Buffer. Mix gently until fully dissolved. Aliquot and store at -20°C.

Reagent D — Signal Development Enzyme Mix

Reconstitute with 220 µL Assay Buffer. Mix gently until fully dissolved. Aliquot and store at -20°C.

Reagent E — ATP Calibrator

Reconstitute with 100 µL deionized water to prepare a concentrated ATP calibrator stock. Keep on ice during use.

Fluorometric Signal Probe Working Solution

For fluorometric detection, prepare a 1:10 diluted Signal Probe working solution before preparing the reaction mix. This improves pipetting accuracy in reduced-volume format.

Reduced-Volume Assay Protocol

This compact format uses 25 µL prepared standard or sample plus 25 µL reaction mix, for a final assay volume of 50 µL per well.

Final well volume: 25 µL standard or sample + 25 µL reaction mix = 50 µL per well.

Sample Preparation

  1. Homogenize or rapidly disrupt approximately 1 × 10⁶ cells or 10 mg tissue.
  2. Use 100 µL of a suitable protein-precipitating solution.
  3. Keep samples cold during preparation.
  4. Centrifuge to remove debris and precipitated proteins.
  5. Transfer the clear supernatant to a clean tube.
  6. Use 2–10 µL prepared sample per well.
  7. Adjust each sample well to 25 µL final sample volume using Assay Buffer.
Important: ATP is unstable. Prepare samples fresh whenever possible. If storage is required, snap-freeze samples rapidly and keep frozen until analysis.

Standard Curve Preparation

Colorimetric Standards

Standard ATP Amount per Well
S0 0 nmol
S1 2 nmol
S2 4 nmol
S3 6 nmol
S4 8 nmol
S5 10 nmol

Fluorometric Standards

Standard ATP Amount per Well
S0 0 pmol
S1 200 pmol
S2 400 pmol
S3 600 pmol
S4 800 pmol
S5 1000 pmol

Reaction Mix Preparation

Prepare enough reaction mix for all standards, samples, and background controls. Include at least 10% extra volume to compensate for pipetting loss.

Colorimetric Reaction Mix

Component Test Reaction Background Control
Assay Buffer 22 µL 23 µL
Signal Probe 1 µL 1 µL
ATP Conversion Enzyme 1 µL —
Signal Development Enzyme Mix 1 µL 1 µL
Total 25 µL 25 µL

Fluorometric Reaction Mix

Component Test Reaction Background Control
Assay Buffer 22 µL 23 µL
Diluted Signal Probe Working Solution 1 µL 1 µL
ATP Conversion Enzyme 1 µL —
Signal Development Enzyme Mix 1 µL 1 µL
Total 25 µL 25 µL

Plate Setup and Measurement

Step Volume or Condition
Standard or sample 25 µL per well
Reaction mix 25 µL per well
Final well volume 50 µL
Incubation Room temperature, protected from light
Colorimetric reading OD 570 nm
Fluorometric reading Ex/Em 535/587 nm

Endpoint signal is typically reached within 30 minutes. Use the same incubation time for standards, samples, and controls.

Typical Colorimetric Results

Representative colorimetric standard curve using ATP standards from 0 to 10 nmol per well.

ATP colorimetric standard curve OD 570 nm
Colorimetric ATP standard curve, OD 570 nm.
ATP Standard Raw OD 1 Raw OD 2 Corrected OD 1 Corrected OD 2
0 nmol 0.041 0.043 0.000 0.000
2 nmol 0.292 0.286 0.251 0.243
4 nmol 0.548 0.556 0.507 0.513
6 nmol 0.806 0.824 0.765 0.781
8 nmol 1.072 1.061 1.031 1.018
10 nmol 1.329 1.345 1.288 1.302
Typical linear fit: OD 570 nm = 0.129 × ATP nmol + 0.002

Typical Fluorometric Results

Representative fluorometric standard curve using ATP standards from 0 to 1000 pmol per well.

ATP fluorometric standard curve Ex Em 535 587 nm
Fluorometric ATP standard curve, Ex/Em 535/587 nm.
ATP Standard Raw RFU 1 Raw RFU 2 Corrected RFU 1 Corrected RFU 2
0 pmol 215 228 0 0
200 pmol 2,840 2,910 2,625 2,682
400 pmol 5,520 5,610 5,305 5,382
600 pmol 8,230 8,145 8,015 7,917
800 pmol 10,780 10,940 10,565 10,712
1000 pmol 13,420 13,310 13,205 13,082
Typical linear fit: RFU = 13.2 × ATP pmol + 18

Calculation

  1. Subtract the blank value from all standards.
  2. Plot corrected signal versus ATP amount.
  3. Determine the slope of the standard curve.
  4. For each sample, subtract the paired background control.
  5. Use the standard curve to calculate ATP amount in the well.

ATP concentration in loaded sample

ATP per µL sample = ATP amount in well ÷ sample volume added

Total ATP in original sample

Total ATP = ATP per µL sample × total recovered sample volume

Normalized ATP content

ATP per mg tissue = total ATP ÷ tissue mass
ATP per cell number = total ATP ÷ number of cells

Technical Notes

  • Use fresh samples whenever possible.
  • Keep samples cold during extraction.
  • Prepare paired background controls for each sample type.
  • Protect detection reagents from light.
  • Use the same incubation time for standards and samples.
  • Verify linearity when using the reduced-volume format.
  • Do not compare colorimetric and fluorometric results without separate standard curves.

Reliable ATP Quantification in a Compact Format

The AffiASSAY® ATP Colorimetric & Fluorometric Assay Kit combines flexible detection, simplified reagent naming, reduced reaction volume, paired background correction, and clear standard-curve-based quantification for routine ATP analysis in research samples.