-
- Target
- D-Lactate
- Application
- Biochemical Assay (BCA)
- Specificity
- 1 μM
- Characteristics
- Sensitive and accurate. Detection limit of 1 µM and linearity up to 50 µM D-lactate in 96-well plate assay. Convenient. The procedure involves adding a single working reagent, and reading the fluorescence after 60 min. Room temperature assay. High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day
- Components
- Assay Buffer: 10 mL. Enzyme A: 120 µL. NAD Solution: 1 mL. Enzyme B: 120 µL. Probe: 750 µL. Standard: 1 m
- Material not included
- Pipetting (multi-channel) devices. Black, flat bottom 96-well plates and fluorescent plate reader capable of reading at ex/em = 530/585 nm.
-
D-Lactate Assay Kit
BCA Cell Culture Supernatant, Plasma, Serum
-
- Application Notes
- Direct Assays: D-lactate in serum, plasma, urine, cell media samples and other biological samples
- Protocol
-
Prepare 1000 µL 40 µM D-lactate Premix by mixing 2 µL 20 mM Standard and 998 µL distilled water. For cell culture samples, prepare 1000 µL 40 µM D-lactate Premix by mixing 2 µL 20 mM Standard and 998 µL culture medium without serum.
Samples. Add 50 µL sample to two separate wells. Set up two reactions for each sample: one with added Enzyme A (Sample) and a No Enzyme A control (Sample Blank). Samples requiring an internal standard, will need three separate reactions: 1) Sample plus Standard, 2) Sample alone and 3) Sample Blank. For the internal standard first prepare 400 µL 250 µM D-lactate standard by mixing 5 µL 20 mM Standard and 395 µL dH2O. For the Sample plus Standard well, add 5 µL 250 µM D-lactate and 45 µL sample. For the Sample and Sample Blank wells, add 5 µL dH2O and 45 µL sample
Reaction: Add 50 µL Working Reagent per reaction well quickly. Tap plate to mix briefly and thoroughly. Incubate for 60 min at RT protected from light. - Reagent Preparation
-
Spin the Enzyme tubes briefly before pipetting. For each Sample and Standard well, prepare Working Reagent by mixing 40 µL Assay Buffer, 1 µL Enzyme A, 1 µL Enzyme B, 10x L NAD and 5 µL Probe. Fresh reconstitution is recommended. For the Sample Blanks, the Working Reagent includes 40 µL Assay Buffer, 1 µL Enzyme B, 10x L NAD and 5 µL Probe (NO Enzyme A).
- Calculation of Results
-
Plot the D-lactate Standard Curve and determine its slope. The D-lactate concentration of the sample is computed as follows: where FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and Sample Blank respectively. Slope is the slope of the standard curve and n is the dilution factor (e.g. n = 3 for serum samples). If an internal standard was needed, the sample D-lactate concentration is computed as follows: where FSAMPLE and FBLANK are the fluorescence intensity values of the Sample and Sample Blank respectively and FSTANDARD is the fluorescence intensity value of the Sample plus Standard.
Note: if the sample F value is higher than the F for 40 µM D-lactate standard or greater than the F for the internal standard, dilute the sample in water and repeat the assay. Multiply the results by the dilution factor - Restrictions
- For Research Use only
-
- Storage
- -20 °C
-
- Target
- D-Lactate
- Background
-
Quantitative determination of D-lactate (lactic acid) by fluorimetric (530/585 nm) method. Procedure: 60 min.
The mitogen-activated protein kinase (MAPK/ERK) pathway plays a key role in cell proliferation, differentiation and migration. Stimulation by mitogens eventually leads to phosphorylation of ERK1 (T202/Y204) and ERK2 (T185/Y187). The MAPK/ERK cascade presents many interesting drug targets for the development of cancer therapies. This cell-based ELISA measures dually phosphorylated ERK1/2 in whole cells. This simple and efficient assay eliminates the need for cell lysate preparation and can be used to study kinase signaling and the effects of kinase inhibitors on cells. In this assay, cells are grown in 96- well plates and treated with ligands or drugs. Cells are then fixed and permeabilized in the wells. ERK1/2 phosphorylation (pERK) is measured using a double immunoenzymatic labeling procedure.
-
You are here:
