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Amplite® Colorimetric Calcium Quantitation Kit *Blue Color*

Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
Calcium dose response was measured on a 96-well black wall/clear bottom plate with the Amplite® Colorimetric Calcium Quantitation Kit. As low as ~ 2.5 µM Ca2+ was detected with 5 minutes incubation time (n=3)
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Fax1-800-609-2943
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Calcium is essential for all living organisms, particularly in cell physiology, where movement of the calcium ion Ca2+ into and out of the cytoplasm functions as a signal for many cellular processes. Calcium is the fifth most abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. Calcium plays an important role in mediating the constriction and relaxation of blood vessels, nerve impulse transmission, muscle contraction, and hormone secretion. The serum level of calcium is closely regulated within a fairly limited range (9 to 10.5 mg/dL) in the human body. Both hypocalcaemia and hypercal caemia are serious medical disorders. Causes of low calcium levels include chronic kidney failure, vitamin D deficiency, and low blood magnesium levels that can occur in severe alcoholism. Amplite® Calcium Detection Kit provides a simple method for detecting calcium in physiology solutions. This kit uses our Calcium Blue™ as the chromogenic calcium indicator. Its absorbance changes in response to calcium binding. Calcium Blue™ binds calcium tightly in the neutral pH range, generating Calcium Blue™-calcium complex that has intense absorption at ~650 nm.

Platform


Absorbance microplate reader

Absorbance600 or 650 nm
Recommended plateClear bottom

Components


Example protocol


AT A GLANCE

Protocol Summary
  1. Prepare test samples and calcium standard solution (50 µL)
  2. Add Calcium Blue™ reagent (50 µL)
  3. Incubate at room temperature for 5-10 minutes
  4. Monitor absorbance intensity at 600 or 650 nm 
Important      Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Calcium standard solution (3 mM)
Add 10 µL of Calcium Standard (300 mM) (Component C) to 990 µL Dilution Buffer (Component B) to get Calcium standard solution (3 mM) and mix well.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/36361


Calcium standard

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of calcium standards and test samples in a clear bottom 96-well microplate. CS = Calcium standard (CS1-CS7); BL = blank control; TS = test sample.
BLBLTSTS
CS1CS1......
CS2CS2......
CS3CS3
CS4CS4
CS5CS5
CS6CS6
CS7CS7
Table 2. Reagent composition for each well
WellVolumeReagent
CS1 - CS750 µLSerial Dilutions
BL50 µLDilution Buffer (Component B)
TS50 µLtest sample
Table 3.
Calcium StandardBlank ControlSerum or Urine
Serial Dilutions: 50 µLDilution Buffer (Compound B): 50 µL50 µL

Calcium assay
  1. Add the serially diluted calcium standards from 150 µM to 2.34 µM into wells from CS1 to CS7 in duplicate.
  2. Add 50 µL of Calcium Blue™ (Component A) to each well of calcium standards, blank control, and test samples to make the total calcium assay volume to 100 µL/well.
    Note     For a 384-well plate, add 25 µL of sample and 25 µL of assay reaction mixture into each well.
  3. Incubate the reaction for 5 to 10 minutes at room temperature, protected from light.
  4. Monitor the absorbance intensity with an absorbance plate reader at OD 600 nm or 650 nm. 

Assay Protocol for Serum and Urine Samples
  1. Take 10 μL of 300 mM Calcium Standard solution (Component C) to 990 µL Dilution Buffer (Component B) to get 3 mM Calcium Standard Solution.
  2. Take 500 μL of 3 mM Calcium Standard Solution to perform 1:2 serial dilutions to get 1.5, 0.75, 0.375, 0.1875, 0.094, 0.047 and 0 mM serially diluted Calcium standards.
  3. Add 10 μL of calcium standard, serum or urine samples and blank control into their respective wells.
  4. Add 200 μL of Calcium Blue™ (Component A) to each well of calcium standard, blank control, and test samples to make the total calcium assay volume of 210 µL/well.
    Note     For a 384-well plate, add 2.5 μL of sample and 50 μL of assay reaction mixture into each well.
  5. Incubate the reactions for 5-10 minutes at room temperature (protected from light).
  6. Measure the absorbance intensities at 600 nm or 650 nm. 

Images


Citations


View all 4 citations: Citation Explorer
The antimicrobial peptide thanatin disrupts the bacterial outer membrane and inactivates the NDM-1 metallo-β-lactamase
Authors: Ma, Bo and Fang, Chao and Lu, Linshan and Wang, Mingzhi and Xue, Xiaoyan and Zhou, Ying and Li, Mingkai and Hu, Yue and Luo, Xiaoxing and Hou, Zheng
Journal: Nature communications (2019): 1--11
Mechanism of triterpenoid saponin mediated augmentation of saporin based immunotoxin cytotoxicity
Authors: Wensley, Harrison James
Journal: (2019)
Microcystin-LR causes sexual hormone disturbance in male rat by targeting gonadotropin-releasing hormone neurons
Authors: Wang, Xueting and Ding, Jie and Xiang, Zou and Jiang, Peipei and Du, Jing and Han, Xiaodong
Journal: Toxicon (2016): 45--55
High efficiency single-step biomaterial-based microparticle fabrication via template-directed supramolecular coordination chemistry
Authors: Lai, Kwok Kei and Renneberg, Reinhard and Mak, Wing Cheung
Journal: Green Chemistry (2016): 1715--1723

References


View all 105 references: Citation Explorer
Calcium, magnesium, iron, zinc and copper concentration in the hair of tobacco smokers
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Journal: Biol Trace Elem Res (2009): 152
Genetic and nongenetic variation in concentration of selenium, calcium, potassium, zinc, magnesium, and phosphorus in milk of Dutch Holstein-Friesian cows
Authors: van Hulzen KJ, Sprong RC, van der Meer R, van Arendonk JA.
Journal: J Dairy Sci (2009): 5754
Comparison of calcium, magnesium, sodium, potassium, zinc, and creatinine concentration in 24-h and spot urine samples in women
Authors: Ilich JZ, Blanusa M, Orlic ZC, Orct T, Kostial K.
Journal: Clin Chem Lab Med (2009): 216
Atomic absorption spectrometry and scanning electron microscopy evaluation of concentration of calcium ions and smear layer removal with root canal chelators
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Journal: J Endod (2009): 727
3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion
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Journal: Toxicol Sci (2008): 97
Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry
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Journal: Naunyn Schmiedebergs Arch Pharmacol (2008): 423
Platelet free cytosolic calcium concentration during ageing of type 2 diabetic patients
Authors: Alex, undefined and ru N, Popov D, Sbarcea A, Amuzescu M.
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Analysis of calcium concentration fluctuations in hepatopancreatic R cells of Marsupenaeus japonicus during the molting cycle
Authors: Zilli L, Schiavone R, Storelli C, Vilella S.
Journal: Biol Bull (2007): 161
Effect of extracts from safflower seeds on osteoblast differentiation and intracellular calcium ion concentration in MC3T3-E1 cells
Authors: Jang HO, Park YS, Lee JH, Seo JB, Koo KI, Jeong SC, Jin SD, Lee YH, Eom HS, Yun I.
Journal: Nat Prod Res (2007): 787
Measurement of serum calcium concentration after administration of four gadolinium-based contrast agents to human volunteers
Authors: Brown JJ, Hynes MR, Wible JH, Jr.
Journal: AJR Am J Roentgenol (2007): 1539