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Pyruvate Assay Kit

Pyruvate (pyruvic acid) is produced by glycolysis. Most notably, pyruvate links glycolysis to cell growth control, energy production, intracellular signaling. Aberrant serum pyruvate levels have been found to correlate with several diseases, including sepsis, motor neuron disease, necrotizing encephalomyelopathy. Thus, it is important to have sensitive assays to evaluate pyruvate concentrations, which can provide valuable clinical and cellular information. Eton's pyruvate assay kit provides a simple and reliable method for quantifying pyruvate in biological samples such as blood (serum and plasma), urine, cells, culture and fermentation supernant, etc. In the assay, pyruvate is oxidized by enzyme reactions to yield color product, which can be measured at 570 nm for colorimetric assay, and the color intensity is proportional to pyruvate contents, therefore the sample pyruvate concentration can be accurately calculated based on the pyruvate stardard curve. The kit is stable until the expiration date under proper storage and handling conditions. The product is for research use only.

Sku #Product NameProduct SizePriceQTY
120004100P Pyruvate Assay Kit-100 Assays w/Reader Plate(1x) 100 assays w/Reader Plate (1x) $260.00 USD
1200041002 Pyruvate Assay Kit 100 assays 100 assays $240.00 USD
 

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Sample Typle

Plasma,Serum,Cell culture supernatant, other body fluid

Components


Method

Colorimetric method at 570nm

Standard Curve

Sensitivity

4µM-200µM

Reaction Time

30 minutes

Applications

For biological research:
Pyruvate measurement in biological samples
For drug/pharm research:
Drug influence on Pyruvate metabolism

Storage

-80°C

Notes

Material needed but not supplied

A plate reader capable of measuring absorbance at 570nm

Adjustable pipettes and a repeat pipettor

Distilled water(milliQ or HPLC-grade)

Clear Flat bottom 96-well plates if not included in the kit purchased

Shipping Icepacks

Reagent Preparation

Note: All kit reagents are frozen. We recommend you spin all vials before opening.

1. Pyruvate Assay Buffer(10x)
Please do not dilute in the provided Pyruvate Assay Buffer bottle itself.

The bottle contains 10ml of 10xPyruvate Assay Buffer. The assay buffer must be equilibrated to room temperature before use. Please make appropriate amount of 1xPyruvate Assay Buffer for the assay, for example, by diluting 1ml of 10xPyruvate Assay Buffer with 9 ml of distilled water. Prepare additional diluted assay buffer if needed. Store at -80˚C.

2. Pyruvate Standard (10x)
Please do not dilutein the provided Pyruvate standard vial itself.

The vial contains 100µl of 2mM Pyruvate Standard. The standard must be equilibrated to room temperature before use. Dilute 100µl of 2mM Pyruvate Standard with 900µl of diluted Pyruvate Assay buffer to prepare a 200μM Pyruvate Standard. 1ml of diluted standard is enough for making 3 standard curves if assayed in duplicate. Store at -80˚C.

3. Assay Probe
The vial contains 45μl of Assay Probe. The assay probe must be equilibrated to room temperature before use. It is sufficient for 100 wells.
Protect from light. Store at -80˚C.

4. Pyruvate Assay Pre-reaction Mix
The vial contains 17μl of Pyruvate Assay Pre-reaction Mix. The pre-reaction mix must be equilibrated to room temperature before use. It is sufficient for 100 wells.Store at -80˚C.

5. Pyruvate Assay Enzyme Mix
The vial contains 65μl of Pyruvate Assay Enzyme Mix. The enzyme mix must be thawed on ice before use. It is sufficient for 100 wells. Thaw the vial on ice.Store at -80˚C.

Protocol

1.Sample Preparation

Blood, Serum, Plasma, other body fluid, or cell culture supernatant can be measured directly by a series of dilutions of the sample (1/2; 1/4; 1/8; …) to ensure the readings are within the standard curve range. Your samples can be diluted with dH2O or diluted Pyruvate Assay Buffer. However, keep in mind if samples (like hemolyzed serum/plasma) contain high level of lactate dehydrogenase capable of converting pyruvate to lactate, it is important for the samples to be deproteinated.

Add 50μl of samples to each well. We suggest that samples be assayed in duplicate.

Note:

If prepared samples are not assayed the same day, store the samples at -80˚C. If samples need to be deproteinated, make sure to deproteinize the samples prior to storing in the freezer. The deproteinated samples will be stable for one month while stored at -80˚C. For frozen samples, dilutions of samples must be done right before assaying.

2.Standard Prepation

Add 50µl, 40µl, 30µl, 20µl, 10µl, 5µl, 1µl, and 0µl of diluted Pyruvate Standard to each well. Then adjust volume to 50μl/well with diluted Pyruvate Assay Buffer.

We suggest that Pyruvate Standards be assayed in duplicate. A standard curve has to be included with samples in each assay.


3.Reaction Solution Preparation

50μl Reaction Solution is required for each well, and 1ml of Reaction Solution is prepared with the followings:
977µlPyruvate Assay Buffer(1x)
15µlPyruvate Assay Enzyme Mix
3µlPyruvate Assay Pre-reaction Mix
8µlAssay Probe

1ml of Reaction Solution is enough for ~20 wells.Prepare appropriate amount of Reaction Solution for the number of assays to be performed.

Note:Please do not keep any leftover Reaction Solution as it has to be made fresh right beforeyou run assays. If old Reaction Solution is used, assays will not work.


4.Perform the assay

a.Add 50 µl of the reaction solution to each well containing sample and standard.

b.Incubate the reaction for 30 minutes in a 37˚C incubator.

c.Measure absorbance at 570 nm in a microplate reader.

5.Calculations

a. Average the OD570 nm values of replicate wells of each Pyruvate Standard, test samples, and blank. Then, correct background by subtracting the value derived from the blank from all sample readings.

b. Make a standard curve by plotting OD570 nm values from each Pyruvate Standards as a function of Pyruvate concentration. This can be done with excel spreadsheet.

c. Calculate the value of Pyruvate in samples using the equation obtained from the linear regression of the standard curve.
Pyruvate (µM)= [ (Corrected absorbance)-(intercept)]/Slope



Material Safety Data Sheet

Date:Oct 28,2015

1. PRODUCT AND COMPANY IDENTIFICATION

1.1 Product identifier

Product name:Pyruvate Assay Kit

Catalog No: 1200041002,120004100P

1.2 Relevant identified uses of the substance or mixture and uses advised against

For research use only.

1.3 Details of the supplier of the safety data sheet

Company:Eton Bioscience Inc

TollFree:1-800-758-1630

Tel:1-800-758-1630

Fax:1-800-507-2912

1.4 Emergency telephone number

1-800-758-1630

2. HAZARDS IDENTIFICATION

2.1 Classification of the substance or mixture

Not a hazardous substance or mixture

2.2 GHS Label elements, including precautionary statements

Not a hazardous substance or mixture

2.3 Other hazards

None

3. COMPOSITION/INFORMATION ON INGREDIENTS

3.1 Substances

Pyruvate Assay Buffer

Pyruvate Enzyme Mix

Components

CAS#

Components

CAS#

Potassium Phosphate Monobasic

7778-77-0

Potassium Phosphate Monobasic

7778-77-0

Potassium Phosphate Dibasic

7758-11-4

Potassium Phosphate Dibasic

7758-11-4

Water

7732-18-5

Pyruvate Oxidase

9001-96-1

Pyruvate Assay Standard

Assay Probe

Components

CAS#

Components

CAS#

Sodium Pyruvate

113-24-6

DMSO

67-68-5

Potassium Phosphate Monobasic

7778-77-0

Pyruvate Assay Pre-reaction Mix

Potassium Phosphate Dibasic

7758-11-4

Components

CAS#

Water

7732-18-5

FAD

84366-81-4

TPP

154-87-0

Water

7732-18-5












4. FIRST AID MEASURES

4.1 Description of first aid measures

Eye contact

Check for and remove contact lenses and flush with copious amounts of water; assure adequate flushing by separating the eyelids with fingers; call a physician

Skin Contact

Flush with copious amounts of water; remove contaminated clothing and shoes; call a physician

Inhalation

Remove to fresh air. If not breathing give artificial respiration ;call a physician

Ingestion

If swallowed, never give anything by mouth to an unconscious person. Rinse mouth with water ;call a physician

4.2 Most important symptoms and effects, both acute and delayed

No information available

4.3 Indication of any immediate medical attention and special treatment needed.

No information available

5. FIRE FIGHTING MEASURES

5.1 Extinguishing media

Suitable extinguishing media

Water spray, alcohol-resistant foam, dry chemical or carbon dioxide

5.2 Special hazards arising from the substance or mixture

No information available

5.3 Advice for firefighters

Wear self-contained breathing apparatus and protective clothing to prevent contact with skin and eyes.

6. ACCIDENTAL RELEASE MEASURES

6.1 Personal precautions, protective equipment and emergency procedures

Use full personal protective equipment. Evacuate personnel to safe areas. Avoid breathing vapors,mist or gas.

6.2 Environmental precautions

Prevent further leakage or spillage. Prevent product from entering drain.

6.3 Methods and material for containment and cleaning up

Keep in suitable, closed containers for disposal

7. HANDLING AND STORAGE

7.1 Precautions for safe handling

Avoid skin/eye contact. Use protective equipment as needed. Wash contaminated clothing before reuse.

7.2 Conditions for safe storage,including any incompatibilities

Keep container tightly closed. Store at -80˚C.

7.3 Specific end use(s)

No data available.

8. EXPOSURE CONTROLS/PERSONAL INFORMATION

8.1 Control Parameters

This product contains no hazardous materials with occupational exposure limits.

8.2 Exposure controls

Engineering controls

Provide shower and eye wash station

Personal protective equipment

Eye Protection

Wear safety goggles

Skin Protection

Wear protective clothing

Hand Protection

Wear protective gloves

Respiratory protection

Wear NIOSH/MSHA approved respirators

9. PHYSICAL AND CHEMICAL PROPERTIES

9.1 Information on basic physical and chemical properties

Pyruvate Assay Buffer

Appearance

Liquid, colorless

pH

No data available

Melting point

No data available

Freezing point

No data available

Boiling point

No data available

Density

No data available

Water solubility

Soluble

Pyruvate Standard

Appearance

Liquid, colorless

pH

7.0

Melting point

No data available

Freezing point

No data available

Boiling point

No data available

Density

No data available

Water solubility

Soluble

Pyruvate Enzyme Mix

Appearance

Liquid, colorless

pH

7.0

Melting point

No data available

Freezing point

No data available

Boiling point

No data available

Density

No data available

Water solubility

Soluble

Assay Probe

Appearance

Liquid, colorless

pH

No data available

Melting point

No data available

Freezing point

No data available

Boiling point

No data available

Density

No data available

Water solubility

Soluble

Pyruvate Pre-reaction Mix

Appearance

Liquid, yellow

pH

No data available

Melting point

No data available

Freezing point

No data available

Boiling point

No data available

Density

No data available

Water solubility

Soluble

9.2 Other safety information

No data available.

10. STABILITY AND REACTIVITY

10.1Reactivity

No data available

10.2Chemical stability

Stable under recommended storage conditions.

10.3Possibility of hazardous reactions

No data available.

10.4Conditions to avoid

No data available

10.5Incompatible materials

Strong oxidizing agents

10.6Hazardous decomposition products

Hazardous decomposition products formed under fire conditions: carbon oxides

11. TOXICOLOGICAL INFORMATION

11.1Information on toxicological effects

Toxicological effects on this product have not been thoroughly studied.

12. ECOLOGICAL INFORMATION

12.1Toxicity

Avoid release into environment

12.2Persistence and degradability

No data available

12.3.Bioaccumulative potential

No data available

12.4Mobility in soil

No data available

12.5Results of PBT and vPvB assessment

No data available

12.6Other adverse effects

No data available

13. DISPOSAL INFORMATION

13.1Waste treatment methods

Dispose in accordance with local, state, and federal regulations.

14. TRANSPORT INFORMATION

DOT:

Proper shipping name: none
Non-Hazardous for transport: this substance is considered to be non-hazardous for transport
IATA:

Proper shipping name: none
Non-Hazardous for transport: this substance is considered to be non-hazardous for transport

ADR/RID

Proper shipping name: none
Non-Hazardous for transport: this substance is considered to be non-hazardous for transport

15. REGULATORY INFORMATION

EU Risk and Safety phrases:

R36/37/38: irritating to eyes/respiratory system/skin

16. OTHER INFORMATION

The information above is believed to be accurate and represents the best information currently available to us. However, we make no warranty of merchantability or any other warranty, express or implied, with respect to such information. Eton Bioscience Inc. shall not be held liable for any damages or other consequences resulting from handling or from contact with the above product.

1. Hansen JL, Freier EF. (1978). Direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin Chem. 24(3):475-9.
2. Howell BF, McCune S, Schaffer R (1979). Lactate-to-pyruvate or pyruvate-to-lactate assay forlactate dehydrogenase: a re-examination. Clin Chem. 25(2):269-72.
3. Arias-Mendoza F, Piña E. (1991). A sensitive multienzymatic assay for the measurement of pyruvate, dihydroxyacetone phosphate, oxaloacetate, and acetoacetate in clear extracts from biological samples. Prep Biochem. 21(4):211-4.
4. Chariot P. et al (1994). Optimal handling of blood samples for routine measurement of lactate and pyruvate. Arch Pathol Lab Med. 118(7):695-7.

Wilkins HM,Harris JL,Carl SM,E L, Lu J,Eva SelfridgeJ,Roy N,Hutfles L,Koppel S,Morris J,Burns JM,Michaelis ML,Michaelis EK,Brooks WM,Swerdlow RH. Oxaloacetate activates brain mitochondrial biogenesis, enhances the insulin pathway, reduces inflammation and stimulates neurogenesis. Hum Mol Genet. 2014 Dec 15;23(24):6528-41. doi: 10.1093/hmg/ddu371. Epub 2014 Jul 15.

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San Diego, CA 92121
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