L-Lactate Assay Kit I

Lactate dehydrogenase converts lactate and NAD+ into pyruvate and NADH. The Lactate Assay Kit is based on the reduction of the terazolium salt INT in a NADH-coupled enzyme reaction to formazan, which exhibits an absorbance maximum at 490 nm. The intensity of the absorbance is proportional to the lactate concentration. Using a set of lactate standards, the assay can measure the concentration of lactate in samples (plasma, medium, or others) in a quantitative manner. The kit is stable until the expiration date under proper storage and handling conditions. The product is for research only.

Product Name L-Lactate Assay Kit I (5 ml)-100 Assays- Assay Solution ONLY
SKU Number 120001100A
Product Size (5ml) - 100 assays- ASSAY SOLUTION ONLY
List Price $98.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (5ml) - 100 assays
SKU Number 1200011002
Product Size (5ml) - 100 assays
List Price $108.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (5 ml)-100 Assays w/Reader Plate (1x)
SKU Number 120001100P
Product Size (5ml) - 100 assays
List Price $120.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (10 ml)-200 Assays- Assay Solution ONLY
SKU Number 120001200A
Product Size (10ml) - 200 assays- ASSAY SOLUTION ONLY
List Price $146.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (10ml) - 200 assays
SKU Number 1200012002
Product Size (10ml) - 200 assays
List Price $156.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (10ml)-200 Assays w/ Reader Plate(2x)
SKU Number 120001200P
Product Size (10ml) - 200 assays
List Price $180.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (20ml) - 400 assays- ASSAY SOLUTION ONLY
SKU Number 120001400A
Product Size (20ml) - 400 assays- ASSAY SOLUTION ONLY
List Price $267.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (20ml) - 400 assays
SKU Number 1200014002
Product Size (20ml) - 400 assays
List Price $277.00 USD
  Quantity    
Product Name L-Lactate Assay Kit I (20ml)-400 Assays w/ Reader Plate(4x)
SKU Number 120001400P
Product Size (20ml) - 400 assays
List Price $326.00 USD
  Quantity    

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Kit Components

SKU No
1200011002
120001100P
1200012002
120001200P
1200014002
120001400P
Assay Solution
1 bottle(5ml)
1 bottle(5ml)
1 bottle(10ml)
1 bottle(10ml)
1 bottle(20ml)
1 bottle(20ml)
Standard1(3.2mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard2(1.6mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard3(0.8mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard4(0.4mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard5(0.2mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard6(0.1mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard7(0.05mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Standard8(0mM)
1 vial
1 vial
1 vial
1 vial
1 vial
1 vial
Flat Bottom Plate
Not included
1 plate
Not included
2 plates
Not included
4 plates
*SKU#120001100A(5ml), 120001200A(10ml), and 120001400A(20ml) provide Assay solution ONLY.

Storage and Handling

1. This kit will perform as described if stored at -80oC and used before its expiration date.
2. If you cannot consume the whole solution in one assay, aliquot and freeze the aliquots at -80 oC.
3. Protect from light.
4. Try to conduct your assay in a dim environment.
5. Do not expose kits to room temperature too long.
6. Do not repeat thaw-freeze cycles.
7. If the above directions are not followed, kits will degrade at a faster rate.

Material needed but not supplied

1. A plate reader capable of measuring absorbance between 470nm-490nm
2. Adjustable pipettes and a repeat pipettor
3. A source of pure water; glass distilled water of HPLC-grade water is sufficient
4. 0.5M Acetic Acid
5. Flat bottom 96-well plates if not included in the kit purchased


Reagent Preparation
1. L-Lactate Standards
For your convenience, eight standards are pre-made and provided with 1000 ul in each tube. They are required to generate a standard curve. The standards should be kept at -80 oC after each use.
2. L-Lactate Assay Solution
The solution contains enzymes that are light sensitive. Best to aliquot the amount needed and use it all to prevent thawing/freezing cycles. Freeze and store aliquots at -80 oC

Assay Protocol
1. Sample Preparation
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; .…..). Solid samples, such as tissues, can be first homogenized and extracted with ethanol (80%) with a tissues/ethanol ratio of 1:8 (1 hr at 4°C) followed by centrifugation at 10,000g. The Clear supernatants then can be measured as described for liquid samples.
2. Standard Curve and Assay Setup
It is recommended that all standards and samples be duplicated in the assay.
a. Thaw L-Lactate assay solution and L-Lactate standards on ice. Add 50ul of each standard per well to the designated wells on the 96-well flat bottom plate.
b. Prepare test samples to a final volume of 50ul/well on the 96-well flat bottom plate. We suggest testing several dilutions of your sample to make sure the readings will fall within the linear range of the standard curve.
3. Perform the Assay
aAdd 50ul of L-Lactate assay solution to each well containing L-Lactate standards and test samples.
b. Incubate for 30mins at 37°C incubator. Note: Please do not use CO2 incubator.
c. Stop the reaction by adding 50 μl of 0.5 M acetic acid per well followed by brief gentle agitation. Note: Eliminate any air bubbles present in the wells using a needle prior to measurement.
d. Measure the absorbance at 490nM using a microplate reader.
4. Calculations
a Average the OD490 nm values of replicate wells of each L-Lactate standard, test samples, and blank. In order to get the corrected absorbance, subtract the average OD490 nm value of the blank (L-Lactate Standard #8) from the average OD490 nm values from all standards and samples.
b Make a standard curve by plotting OD490 nm values from each L-Lactate standards as a function of L-Lactate concentration. This can be done with excel spreadsheet. Calculate the value of L-Lactate in samples using the equation obtained from the linear regression of the standard curve.
L-Lactate(uM)= [ (Corrected absorbance)-(y-intercept)]
Slope
The standard curve below is an example of the data typically provided with this kit. However, your results mayvary from this. Please do not use this data to determine the values of your sample. You will need to run a new standard curve each time.

MATERIAL SAFETY DATA SHEET
TRADE NAME: L-Lactate Assay Kit I
DATE OF ISSUE:March 10,2014
SKU#: 12001100A.1200011002, or 120001100P(100 assays), 120001200A,1200012002, or 120001200P (200 assays), 120001400A.1200014002, or 120001400P (400 assays)

TRADE NAME:L-Lactate Assay Kit I
Manufacturer: Eton Bioscience Inc., 5820 Oberlin Drive, Suite 108, San Diego, CA 92121
Information Telephone:800.758.1630
Emergency Telephone: 800.758.1630

SECTION II: COMPOSITION / INFORMATION ON INGREDIENTS

CHEMICAL CHARACTERIZATION:
L-Lactate Assay Kit Solution:
CAS#

L-Lactate Assay Standard
CAS#
DMSO
67-68-5

Sodium L-Lactate
867-56-1
BSA
9048-46-8

Tris-HClpH7.5
N/A
Beta-NAD
53-84-9

Water
7732-18-5
INT
146-68-9



L-LDH
9001-60-9



PMS
299-11-6



Tris-HCl pH 7.5
N/A



Water
7732-18-5



SECTION III:HAZARD IDENTIFICATION

Potential harmful if any component is swallowed. May cause irritation if contacted with skin or eyes. In such a case, flush skin or eyes with water for 15 min. Get medical attention if necessary

SECTION IV:FIRST AID MEASURES

General Information: Remove and wash contaminated clothing promptly.
IN CASE OF INHALATION:Supply fresh air and then consult a doctor.
IN CASE OF EYE CONTACT:Immediately flush eyes with copious amounts of water for 15 minutes.
IN CASE OF SKIN CONTACT:Immediately wash skin with soap and copious amounts of water.
IF SWALLOWED:Rinse mouth with copious amounts of water. Then consult a doctor.

SECTION V:FIREFIGHTING MEASURES

EXTINGUISHING AGENTS:Noncombustible.

SECTION VI:ACCIDENTAL RELEASE MEASURES

PRECAUTIONARY MEASURES:No special measures required.
CLEAN-UP PROCEDURES:No special measures required.

SECTION VII:HANDLING AND STORAGE

HANDLING:
Exposure Limit: Undetermined;
Toxicity: We are not aware of any toxicity data for this product;
Carcinogenicity: Unknown;
Exposure symptoms: Unkown, handle with care;
Handling: Gloves, protective clothing and eyewear should be worn and safe laboratory practices followed;
In case of spill: Use appropriate protective equipment and methods to clean up spilled substance promptly. Absorb onto an appropriate material. Collect and dispose of all waste in accordance with applicable laws.
STORAGE: Keep tightly closed. Store at -80oC freezer.

SECTION VIII:EXPOSURE CONTROLS / PERSONAL PROTECTION

The routine precautionary measures for handling chemicals should be followed such as wearing chemical safety goggles, rubber gloves, rubber boots, NIOSH / MSHA-Approved respirator when handling chemicals and wash thoroughly after handling.Handle chemicals with care and avoid contact with eyes, skin, or on clothing.Use safety shower and eye bath as first aid measures if exposed to chemicals.

SECTION IX:PHYSICAL AND CHEMICAL PROPERTIES

PHYSICAL STATE / FORM:Liquid
COLOR:Yellowish
ODOR:None
pH FACTOR:(20°C)around 7
VISCOSITY:(20°C)N/A
MELTING POINT:N/A
BOILING POINT:N/A
IGNITION TEMPERATURE:N/A
FLASHPOINT:N/A
EXPLOSION LEVEL:LOWER: N/A
UPPER: N/A
VAPOR PRESSURE: N/A
SPECIFIC GRAVITY: N/A
SOLUBILITY IN WATER: Soluble

SECTION X:STABILITY AND REACTIVITY

SUBSTANCES TO BE AVOIDED: N/A
DANGEROUS REACTIONS: No dangerous reactions known.
HAZARDOUS, COMBUSTION, OR DECOMPOSITION PRODUCTS:No dangerous decomposition products known.

SECTION XI:TOXICOLOGICAL INFORMATION

TOXICITY DATA:N/A
ACUTE EFFECTS INHALATION:N/A.
EYE CONTACT:May cause eye irritation.
SKIN CONTACT:May cause skin irritation.
INGESTION:May be harmful if swallowed.
PROLONGED EXPOSURE: N/A
CHRONIC EFFECTS: N/A
RTECS number:N/A
ADDITIONAL INFORMATION:The product should be handled with routine precautionary measures for handling chemicals.Additional harmful properties cannot be ruled out.

SECTION XII:ECOLOGICAL INFORMATION

No information available.

SECTION XIII:DISPOSAL CONSIDERATIONS

Chemical waste generators must determine whether a discarded chemical is classified as a hazardous waste. US EPA guidelines for the classification determination are listed in 40 CFR Parts 261.3. Additionally, waste generators must consult state and local hazardous waste regulations to ensure complete and accurate classification.

SECTION XIV:TRANSPORT INFORMATION

DOT REGULATIONS: Not applicable.

SECTION XV:REGULATORY INFORMATION

No information available.

SECTION XVI: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, and we assume no liability resulting from its use. Users should make their own investigations to determine the suitability of the information for their particular purposes. Eton Bioscience Inc. shall not be held liable for any damages or other consequences resulting from handling or from contact with the above product.


Q. What type of medium should I use for making cultured cells for Lactate assay?
A. Please use phenol red free medium. Please do not use phenol red medium since phenol red would affectabsorbance readings.

Q. What enzymes is used in Lactate Assay?
A. It is LDH.

Q. Can you tell me the compositions of your Lactate Assay?
A. Unfortunately it is proprietary.

Q. What type of anticoagulant should I use for my samples?
A.If you are collecting blood for measuring lactate from serum ,you do not have to collect blood using an anticoagulant.If you are collecting blood for measuring lactate from blood, you can use heparin or EDTA.

Q. How do I know whether I need to use L-Lactate or D-Lactate assay kit?
A. L-Lactate assay kits are commonly used for measuring lactate level in animal cells.
D-lactate assay kits are commonly used for measuring lactate level in bacteria cells.

Q. Do I need to make a standard curve everytime?
A. Yes, it is necessary since you calculate your sample concentration based on the standard curve. Please do not usethe representative Lactate standard curve in the protocol to calculate concentrations of your samples.

Q.Can I use fluorescence spectroscopy to measure readings for Lactate assay kits?
A. No, the kit does not work with a fluorescence reader since the kit employs colorimetric assays.

Q. How do I measure Lactate from cultured cells?
A. You can grow cells in phenol red-free medium, then you can extract cells with 80% ethanol.

Q. Can I use frozen samples?
A. Although it is better to use fresh samples for assays, you canuse frozen samples. If you are not using frozen samples in once, please make aliquots of your samples before you put them in a freezer to prevent from degrading samples from repeated freeze-thaw cycles.
Q. My sample formed preciptation after adding acetic acid. How can I prevent this?
A. You can still measure readings without adding acetic acid.
Q. The protocol recommends measuring the absorbance at 490nm. Is 485nm close enough?
A. It is ok as long as you set up tje absorbance between 470 and 490nm.
Q. Will L-Lactate assay measure D-Lactate?
A. No, it wont.
Q. Can I use a transparent wall 96-well plate with Lactate Kit?
A. Yes, you can.

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2. Jun Mifune, Katrin Grage, and Bernd H. A. Rehm.:Production of Functionalized Biopolyester Granules by Recombinant Lactococcus lactis.Appl. Envir. Microbiol., Jul 2009; 75: 4668 - 4675.


3. Dragutin J. Savic, William M. McShan, and William M. McShan.:Long-term survival of Streptococcus pyogenes in rich media is pH-dependent.Microbiology, Jun 2012; 158: 1428 - 1436


4 Kristin A. Anderson, Fumin Lin, Thomas J. Ribar, Robert D. Stevens, Michael J. Muehlbauer, Christopher B. Newgard, and Anthony R. Means.:Deletion of CaMKK2 from the Liver Lowers Blood Glucose and Improves Whole-Body Glucose Tolerance in the Mouse.Mol. Endocrinol., Feb 2012; 26: 281 - 291.


5.Janina P. Lewis, Divya Iyer, and Cecilia Anaya-Bergman.:Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate.Microbiology, Nov 2009; 155: 3758 - 3774.


6. Amparo Wolf, Sameer Agnihotri, Johann Micallef, Joydeep Mukherjee, Nesrin Sabha, Rob Cairns, Cynthia Hawkins, and Abhijit Guha.:Hexokinase 2 is a key mediator of aerobic glycolysis and promotes tumor growth in human glioblastoma multiforme.J. Exp. Med., Feb 2011; 208: 313 - 326.


7.Inna Serganova, Asif Rizwan, Xiaohui Ni, Sunitha B. Thakur, Jelena Vider, James Russell, Ronald Blasberg, and Jason A. Koutcher.:Metabolic Imaging: A Link between Lactate Dehydrogenase A, Lactate, and Tumor Phenotype.Clin. Cancer Res., Oct 2011; 17: 6250 - 6261.


8.Changlu Liu, Jiejun Wu, Jessica Zhu, Chester Kuei, Jingxue Yu, Jonathan Shelton, Steven W. Sutton, Xiaorong Li, Su Jin Yun, Taraneh Mirzadegan, Curt Mazur, Fredrik Kamme, and Timothy W. Lovenberg.:METABOLISM, REGULATION, AND SIGNALING: Lactate Inhibits Lipolysis in Fat Cells through Activation of an Orphan G-protein-coupled Receptor, GPR81.J. Biol. Chem., Jan 2009; 284: 2811 - 2822.


9.Marina Ostroukhova, Nicholas Goplen, Md Zunayet Karim, Lidia Michalec, Lei Guo, Qiaoling Liang, and Rafeul Alam.:The role of low-level lactate production in airway inflammation in asthma.Am J Physiol Lung Cell Mol Physiol, Feb 2012; 302: L300 - L307.



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San Diego, CA 92121
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