Download L-LACTATE Assay Kit KC-04-001 (Fluorometric) 100 tests (96 well

L-LACTATE Assay Kit
KC-04-001 (Fluorometric)
100 tests (96 well plate)
Index
Introduction
Materials
Assay Principle
Assay protocol
Data analysis
References
Warranties and Limitation of Liability
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Introduction
Lactic Acid is an alpha hydroxyl acid that can ionize a carboxyl proton to
originate the lactate ion, this ion subsists as two optical isomers L-Lactate
and D-Lactate. The enzyme lactate dehydrogenase catalyzes the change
method of pyruvate to lactate in animals during the phase of fermentation1.
Blood levels of lactate can fluctuate between 1 and 20 mM, depending on
the levels of exercise2.
Exceptionally elevate concentrations of lactate have been correlated to
illness states such as diabetes and lactate acidosis. L(+)-Lactate is the
most stereo-isomer of lactate produced in human intermediary metabolism
and exists in blood. In addition, D(-)-Lactate isomer exist but just between
1-5% of the concentration of L(+)-Lactate.
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Materials
Bioquochem Lactate Assay Kit contains:
Product
Quantity
Lactate Reagent A*
Lactate Reagent B*
Lactate Reagent C*
Lactate Reagent D
Lactate Standard
2 vials
2 vials
1 vial
2 vials
1 vial
Storage
-20 ºC
-20 ºC
-20 ºC
4 ºC
4 ºC
*These reagents are stable during 10 days at 4ºC and is shipped in these conditions. Once received keep
them at -20ºC.
 This kit is for R&D use only
All these chemicals should be handled with care
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Assay principle
In the Bioquochem L-Lactate Assay Kit, lactate particularly operates with
an enzyme mix to create a product. This one, interacts with lactate probe
to generate a fluorescence signal (Ex/Em = 535/587 nm)3. The kit is an
adequate tool to detect L (+)-Lactate in many biological samples (blood,
culture cells, etc.). In this kit is not necessary a previous treatment of the
samples. This Kit is able to detect lactate in the range of 1 nmol.
Each kit supplies enough quantity of reagents to asses 100 tests.
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Assay Protocol
Standard Preparation
For the fluorometric assay it is necessary to perform a calibrate curve
following the Table 1.
Table 1. Reagent volumes needed to carry out the standard curve
Sample
S1(Blank)
S2
S3
S4
S5
S6
S7
Standard
Reagent D
Lactate
(µl)
(µl)
(nmol/ well)
0
8
16
24
32
40
48
200
192
184
176
168
160
152
0
2
4
6
8
10
12
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Assay Protocol
Plate set up
Fill the plate following the indication in Figure 1.
Figure 1. 96-well plate filling format
S1-S7 = Standards
C0-C40 = Samples
Attention
 This scheme is just a recommendation of how to perform the assay.
 For optimal results, it is recommended to run the standards and the samples
for duplicate, but it is the user´s discretion to do so.
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Assay Protocol
Performing the assay
1. In order to ensure the reading are within the standard curve range we
recommend to add 2-50 µl test samples (50 µl in case of standard) to
a 96-well sample and to adjust the volume to 50 µl/ well with reagent
D.
2.
Add 90 µl of Reagent A to the plate
3.
Add 50 µl Reagent B to the plate
4.
Add 10 µl Reagent C to the plate
5.
Keep the sample shaking in the darkness during 30 minutes.
6.
Read the fluorescence at Ex/Em = 535/587 nm.
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Data analysis
Calculation: Subtract Blank Standard reading from all readings. Plot the
Lactate Standard Curve. Apply the corrected OD to the Lactate Standard
Curve to get concentration (nmol) of Lactate in the sample well.
Lactate concentration (mM) = B/V x D
Where:
B is the amount of Lactate in the sample well (nmol)
V is the sample volume added into the reaction well (µl)
D is the sample dilution factor
Figure 2. L-Lactate standard curve
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References
1. R. Tienhaara, J. E. Meany. Lactate dehydrogenase catalyzed reduction
of pyruvate. Active substrate and substrate inhibition. Biochemistry,
1973, 12 (11), pp 2067–2070.
2. Goodwin ML1, Harris JE, Hernández A, Gladden LB. Blood lactate
measurements and analysis during exercise: a guide for clinicians. J
Diabetes Sci Technol. 2007 Jul;1(4):558-69.
3. Mahato B1, Home P, Rajendran G, Paul A, Saha B, Ganguly A, Ray S,
Roy N, Swerdlow RH, Paul S. Regulation of mitochondrial function and
cellular energy metabolism by protein kinase C-λ/ι: a novel mode of
balancing pluripotency. Stem Cells. 2014 Nov;32(11):2880-92.
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Warranties and Limitation of Liability
Bioquochem shall not in any event be liable for incidental, consequential
or special damages of any kind resulting from any use or failure of the
products, even if Bioquochem has been advised of the possibility of such
damage including, without limitation, liability for loss of use, loss of work in
progress, down time, loss of revenue or profits, failure to realize savings,
loss of products of buyer or other use or any liability of buyer to a third party
on account of such loss, or for any labor or any other expense, damage or
loss occasioned by such product including personal injury or property
damage is caused by Bioquochem´s gross negligence. Any and all liability
of Bioquochem hereunder shall be limited to the amounts paid by buyer for
product.
Buyer´s exclusive remedy and Bioquochem´s sole liability hereunder shall
be limited to a refund of the purchase price, or the replacement of all
material that does not meet our specifications.
Said refund or replacement is conditioned on buyer giving written notice to
Bioquochem within 30 days after arrival of the material at its destination.
Expiration date: 1 year from the date of delivery
For further details, please refer to our website www.bioquochem.com.
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