Product Manual

Total Phosphatidic Acid Assay Kit
Catalog Number
MET-5019

100 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Phosphatidic Acid (PA) is a critical precursor for the biosynthesis of many lipids in the cell. PA plays acritical role in membrane structure and acts as a signaling lipid, recruiting cytosolic proteins to the cell
membrane. Within the cell, PA concentrations are maintained at extremely low levels by the activity ofpotent phospholipid phosphatases, converting phosphatidic acid to diacylglycerol (DAG). Because
DAG is another important lipid precursor, it too is rapidly metabolized into other membrane lipid
components (e.g., phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine). Ultimately,
measurement of cellular PA is useful for monitoring lipid synthesis and metabolism.
Cell Biolabs’ Total Phosphatidic Acid Assay Kit measures total phosphatidic acid content, including
lysophosphatidic acid (LPA), in samples by a coupled enzymatic reaction system. First, lipase is usedto hydrolyze phosphatidic acid in samples to glycerol-3-phosphate. Next, the glycerol-3-phosphateproduct is oxidized by glycerol-3-phosphate oxidase (GPO), producing hydrogen peroxide which reacts
with the kit’s Fluorometric Probe (Ex. 530-560 nm/Em. 585-595 nm).

The Total Phosphatidic Acid Assay Kit is a simple, fluorometric assay that quantitatively measures total
PA (PA and LPA) in samples using a 96-well microtiter plate format. Each kit provides sufficient
reagents to perform up to 100 assays, including blanks, standards and unknown samples. The kit
contains an L-α-Phosphatidic Acid Standard and has a detection sensitivity limit of ~5 µM.
Note: This kit is not intended for urine, plasma, or serum samples.

Related Products
1.
2.
3.
4.
5.

STA-397: Serum Triglyceride Quantification Kit (Fluorometric)
STA-399: Free Glycerol Assay Kit (Fluorometric)
STA-600: Phosphatidylcholine Assay Kit
STA-619: Free Fatty Acid Assay Kit (Fluorometric)
MET-5129- Lysine Assay Kit (Fluorometric)

Kit Components
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2.
3.
4.
5.

Phosphatidic Acid Standard (Part No. 50191D): One 200 µL vial of 1 mM L-α-Phosphatidic Acid.
10X Assay Buffer (Part No. 50192D): One 1.5 mL vial.
Lipase Solution (Part No. 50193D): Three 1.4 mL vials.
Enzyme Mixture (Part No. 50194D): Three 1.75 mL vials.
Fluorometric Probe (Part No. 261901): One 110 µL amber vial.

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Materials Not Supplied
1. Standard 96-well fluorescence black microtiter plate
2. PBS (containing Magnesium and Calcium)
3. Extraction reagents for cellular lipids (methanol, chloroform, 1M NaCl)

Storage
Store the entire kit at -80ºC. Avoid multiple freeze/thaws by aliquoting. The Fluorometric Probe islight sensitive and should be maintained in amber tubes.

Preparation of Reagents•••••Phosphatidic Acid Standard: Thaw at room temperature. Once homogeneous and mixed well,maintain the standard at room temperature during assay preparation. The solution is stable for 1
week at room temperature. For longer term storage, the solution should be aliquoted and frozen at 80ºC to avoid multiple freeze/thaws.
1X Assay Buffer: 10X Assay Buffer should be thawed/maintained at 4ºC during assay preparation.
Dilute the 10X Assay Buffer with deionized water. Stir to homogeneity. The 1X solution is stablefor 1 month at 4ºC. For longer term storage, any unused 10X stock material should be aliquoted andfrozen at -80ºC to avoid multiple freeze/thaws.
Lipase Solution and Enzyme Mixture: Thaw at 4ºC. Once homogeneous and mixed well, maintainthe solution at 4ºC during assay preparation. The solution is stable for 1 week at 4ºC. For longer
term storage, the solution should be aliquoted and frozen at -80ºC to avoid multiple freeze/thaws.
Note: These components are provided in multiple tubes to minimize multiple freeze/thaws.
Fluorometric Probe: Thaw and maintain at room temperature during assay preparation. Any unusedmaterial should be aliquoted and frozen at -80ºC to avoid multiple freeze/thaws.
PEU (pre-equilibrated upper phase) Solution: Mix 50 mL of chloroform, 50 mL of methanol, and45 mL of 1M NaCl in a glass container. Shake or mix the solution well, then allow it to separate
into 2 phases. Use the upper phase for washing during the extraction.

Preparation of Phosphatidic Acid Standard
Thaw the Phosphatidic Acid Standard at room temperature. Mix well by vortexing to ensure thesolution is homogeneous. Freshly prepare a dilution series of standard in the concentration range of 0µM – 250 µM by diluting the standard stock solution (provided at 1 mM) in 1X Assay Buffer (seeTable 1). Standards should be prepared fresh, vortexed well and used immediately.
Standard Tubes
123456781 mM Phosphatidic Acid
Standard (μL)
2550 of Tube #150 of Tube #250 of Tube #350 of Tube #450 of Tube #550 of Tube #601X Assay Buffer(µL)
7550505050505050

Table 1. Preparation of Phosphatidic Acid Standards

3Final Phosphatidic AcidStandard (μM)
250
125
62.5
31.2
15.6
7.8
3.9
0

Preparation of Samples••

Urine, plasma and serum: This kit is not recommended for these samples.
Cell Lysates:For adherent cells, remove media and wash cells twice with cold PBS. Harvest ~1x 107 cells byusing a rubber policeman. Do not use proteolytic enzymes. Centrifuge at 1500 x g for 10 minutes.
Carefully remove the supernatant and resuspend in 1 mL of cold PBS. Proceed to step 1 of theextraction procedure below.
For suspension cells, collect ~1 x 107 cells by centrifugation at 1500 x g for 10 minutes. Carefully
remove the supernatant and wash the cell pellet with cold PBS. Repeat PBS wash/centrifugation
once more. Carefully discard the supernatant and resuspend in 1 mL of cold PBS. Proceed to step
1 of the extraction procedure below:
Extraction Procedure
1. Sonicate the 1 mL of cell suspension on ice.
2. Add 1.5 mL of methanol to the sonicated sample.
3. Add 2.25 mL of 1 M NaCl and 2.5 mL of chloroform to the sample. Vortex well.
4. Centrifuge at 1500 x g for 10 minutes at 4ºC to separate the phases.
5. Carefully remove the upper aqueous phase and discard.
6. Wash the lower chloroform phase 2 times with 2 mL of pre-equilibrated upper phase (PEU) (seePreparation of Reagents Section). Separate the phases each time by centrifuging at 1500 x g for
10 minutes at 4ºC. Carefully remove the upper phase and discard each time.
7. After the final wash, carefully transfer the lower organic phase to a glass vial or tube (a syringe
works well). Avoid transferring any remaining upper, aqueous phase.
8. Dry the lower phase in a speedvac or under a gentle stream of nitrogen.
9. Resuspend the dried sample with 50 µL of 1X Assay Buffer (1:20 of the original volume).
Samples may be stored at -80ºC for up to a month.

Assay Protocol
Note: Each PA standard and sample should be assayed in duplicate or triplicate. A freshly preparedstandard curve should be used each time the assay is performed. Maintain the Lipase Solution andEnzyme Mixture at 4ºC during assay preparation.
1. Add 10 µL of the PA standards, samples or blanks to the 96-well microtiter plate.
2. Add 40 µL of Lipase Solution to each well.
3. Incubate at 37ºC for 30 minutes.
4. During the step 3 incubation, separately prepare the desired volume of Detection Enzyme
Mixture according to Table 2, based on the number of tests to be performed. Maintaining allcomponents and mixtures at 4ºC throughout this step, add components in the following
sequence:a. In a tube, add the appropriate volume of Enzyme Mixture.
b. To the Enzyme Mixture, add the corresponding volume of Fluorometric Probe. Mix welland immediately use.
4Note: Detection Enzyme Mixture may appear slightly pink in color. This is normal background andshould be subtracted from all absorbance values (see step 9 for calculation).
EnzymeFluorometricMixture (mL) Probe (µL)
550
2.525
1.25
13Total Volume of Detection
Enzyme Mixture (mL)
5.05
2.525
1.263

# of Tests in 96-well
Plate (100 µL/test)
10050
25

Table 2. Preparation of Detection Enzyme Mixture

5.
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8.

Transfer 50 µL of the above Detection Enzyme Mixture (from step 4) to each well.
Cover the plate wells to protect the reaction from light.
Incubate at room temperature for 10 minutes.
Read the plate with a fluorescence microplate reader equipped for excitation in the 530-560 nm
range and for emission in the 585-595 nm range.
9. Calculate the concentration of phosphatidic acid within samples by comparing the sample
fluorescence to the standard curve. Negative controls (without PA) should be subtracted fromeach PA sample and standard.

Example of Results
The following figures demonstrate typical Total Phosphatidic Acid Assay Kit results. One should use
the data below for reference only. This data should not be used to interpret actual results.

Figure 1: Total Phosphatidic Acid Assay Standard Curve. PA standard curve was performed according to theAssay Protocol. Background has been subtracted.

References
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8.

Wang, X. et al. (2006) Prog. Lipid Res. 45, 250-278.
Merida, I. et al. (2008) Biochem. J. 409, 1-18.
Jenkins, G.M. and Frohman, M.A. (2005) Cell Mol. Life Sci. 62, 2305-2316.
Foster, D.A. and Xu, L. (2003). Mol. Cancer Res. 1, 789-800.
Fang, Y. et al. (2001) Science 294, 1942-1945.
Avila-Flores, A. et al. (2005). J. Biol. Chem. 280, 10091-10099.
Schmelzle, T. and Hall, M.N. (2000) Cell 103, 253-262.
Lim, H.K. et al. (2003) J. Biol. Chem. 278, 45117-45127.
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Recent Product Citations
1. Liang, B. et al. (2021). TBX3 functions as a tumor suppressor downstream of activated CTNNB1mutants during hepatocarcinogenesis. J Hepatol. doi: 10.1016/j.jhep.2021.01.044.
2. Uji, T. et al. (2022). Phospholipase D activation is required for 1-aminocyclopropane 1-carboxylic
acid signaling during sexual reproduction in the marine red alga Neopyropia yezoensis(Rhodophyta). BMC Plant Biol. 22(1):181. doi: 10.1186/s12870-022-03575-z.
3. Nath, A.S. et al. (2022). Modulation of the cell membrane lipid milieu by peroxisomal β-oxidationinduces Rho1 signaling to trigger inflammatory responses. Cell Rep. 38(9):110433. doi:
10.1016/j.celrep.2022.110433.
4. Li, R.H. et al. (2021). A phosphatidic acid-binding lncRNA SNHG9 facilitates LATS1 liquid-liquid
phase separation to promote oncogenic YAP signaling. Cell Res. doi: 10.1038/s41422-021-00530-9.
5. Nadeem, A. et al. (2021). Phosphatidic acid-mediated binding and mammalian cell internalizationof the Vibrio cholerae cytotoxin MakA. PLoS Pathog. 17(3):e1009414. doi:
10.1371/journal.ppat.1009414.
6. Loh, J. et al. (2019). An acute decrease in plasma membrane tension induces macropinocytosis via
PLD2 activation. J Cell Sci. 132(17). pii: jcs232579. doi: 10.1242/jcs.232579.
7. Kumar Sah, R. et al. (2019). Phosphatidic acid homeostasis regulated by a type-2 phosphatidic acidphosphatase represents a novel druggable target in malaria intervention. Cell Death Discov. 5:107.
doi: 10.1038/s41420-019-0187-1.
8. Zhang, P. et al. (2019). Lipin 2/3 phosphatidic acid phosphatases maintain phospholipid
homeostasis to regulate chylomicron synthesis. J Clin Invest. 129(1):281-295. doi:
10.1172/JCI122595.
9. Han, H. et al. (2018). Regulation of the Hippo Pathway by Phosphatidic Acid-Mediated LipidProtein Interaction. Mol Cell. 72(2):328-340.e8. doi: 10.1016/j.molcel.2018.08.038.
10. Lutkewitte, A.J. et al. (2018). Lipin deactivation after acetaminophen overdose causes phosphatidic
acid accumulation in liver and plasma in mice and humans and enhances liver regeneration. FoodChem Toxicol. 115:273-283. doi: 10.1016/j.fct.2018.03.014.

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exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products. In
no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

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