L-Lactate Assay Kit,IC-6570

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L-Lactate Assay Kit  provides a convenient means for detecting L(+)-Lactate in biological samples such as blood, cells, culture mediums, fermentation mediums, etc. There is no need for pretreatment or purification of samples.

In the lactate assay protocol, lactate specifically reacts with an enzyme mix to generate a product,which interacts with a lactate probe to produce color (570 nm) and fluorescence (at Ex/Em =535/587 nm).

Lactate (CH3CH(OH)COO-) plays important roles in many biological processes. Abnormal high concentration of lactate has been related to disease states such as diabetes and lactate acidosis, etc. L(+)-Lactate is the major stereoisomer of lactate formed in human intermediary metabolism and is present in blood. The lactate to pyruvate ratio reflects the redox state of the celland describes the balance beween NAD and NADH, which is dependent on the interconversion of lactate and pyruvate via lactate dehydrogenase (LDH).

Materials Not Supplied

These materials are not included in the kit, but will be required to successfully utilize this assay:

• PBS

• Microcentrifuge

• Pipettes and pipette tips

• Colorimetric or fluorescent microplate reader ¨C equipped with filter for OD570 nm or

Ex/Em = 535/587 nm (respectively)

• 96 well plate: black plates (clear bottoms) for fluorometric assay; clear plates for

colorimetric assay

• Orbital shaker

• Dounce homogenizer (if using tissue)

• If performing deproteinization step, additional reagents are required:

• Perchloric acid (PCA) 4M, ice cold

• Potassium Hydroxide (KOH) 2M

• 10 kD Spin Columns (ab93349) ¨C for fluid samples, if not performing PCA precipitation

Usage

1. Harvest the amount of cells necessary for each assay (initial recommendation = 2 x 106 cells).

2. Wash cells with cold PBS.

3. Resuspend the cell pellet in 4x volumes of Assay Buffer II/Lactate Assay Buffer (~200 ¦ÌL).

4. Homogenize cells quickly by pipetting up and down a few times.

5. Centrifuge 2 ¨C 5 minutes at 4¡ãC at top speed in a cold microcentrifuge to remove any insoluble material.

6. Collect supernatant and transfer to a clean tube.

7. Keep on ice.

8. Cell samples may contain endogenous LDH that will degrade lactate. Remove enzyme from sample by using Deproteinizing Sample Preparation Kit ¨C TCA. Alternatively, you can perform a PCA/KOH deproteinization step following the protocol described below.

Storage

Store at -20¡ãC.

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