This kit may be used to measure the liberation of free orthophosphate in various applications, including Nucleoside Triphosphatase Assays, which involve the liberation of phosphate from nucleoside triphosphates such as ATP, GTP, TTP, and CTP.
MAK307
Malachite Green Phosphate Assay Kit
sufficient for 2500 colorimetric tests
Synonym(s):
Phosphate Determination Assay Kit
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About This Item
NACRES:
NA.84
UNSPSC Code:
12161503
Pricing and availability is not currently available.
usage
sufficient for 2,500 colorimetric tests
packaging
pkg of 1 kit
storage condition
dry at room temperature
technique(s)
activity assay: suitable, protein quantification: suitable
input
sample(s)
detection method
colorimetric
relevant disease(s)
cardiovascular diseases; gastrointestinal diseases
storage temp.
2-8°C
General description
Phosphate is one of the most abundant minerals in the human body and contains about 1 % of total body weight. Phosphate homeostasis is maintained by the several regulators such as parathyroid hormone (PTH), calcitriol and phosphatonins. Maintenance of phosphate homeostasis is essential for various biological processes, such as energy metabolism, cell signaling, regulation of protein synthesis, skeletal development and bone integrity.† An adequate level of phosphate is needed for the process of apoptosis of mature chondrocytes in the growth plate. Phosphate deficiency is associated with the bone pathology and clinical illness.†
Application
Suitable for phosphate determination in a variety of biological and environmental samples.
Malachite Green Phosphate Assay Kit has been used in phosphate quantification and steady-state kinetics.
Malachite Green Phosphate Assay Kit has been used to measure the liberation of free orthophosphate in the following applications:
- Phosphatase Assays: liberation of phosphate from peptide, protein, or small molecule substrate.
- Lipase Assays: liberation of phosphate from phospholipids
- Nucleoside Triphosphatase Assays: liberation of phosphate from nucleoside triphosphates (ATP, GTP, TTP, CTP etc).
- Quantitation of phosphate in phospholipids, proteins and DNAs, etc.
- Drug Discovery: high-throughput screen for phosphatase inhibitors.
Biochem/physiol Actions
Malachite Green is a basic dye belonging to the triphenylmethane group. It is used in biology for the staining of microscopic samples, such as plant tissue, yeast and bacteria. The malachite green method is a sensitive method for the detection of released phosphate.†† The Malachite Green Phosphate Assay Kit is based on quantification of the green complex formed between Malachite Green, molybdate and free orthophosphate. The rapid color formation from the reaction can be conveniently measured on a spectrophotometer (600 - 660 nm) or on a plate reader. The non-radioactive colorimetric assay kits have been optimized to offer superior sensitivity and prolonged shelf life. The assay is simple and fast, involving a single addition step for phosphate determination. Assays can be executed in tubes, cuvettes or multi-well plates. The assays can be conveniently performed in 96 and 384 well plates for high throughput screening of enzyme inhibitors.
Features and Benefits
Compatible with high-throughput handling systems.
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This Item | |||
|---|---|---|---|
| technique(s) activity assay: suitable, protein quantification: suitable | technique(s) - | technique(s) microbe id | staining: suitable | technique(s) microbe id | staining: suitable |
| detection method colorimetric | detection method colorimetric | detection method - | detection method - |
| usage sufficient for 2,500 colorimetric tests | usage sufficient for 500 colorimetric tests | usage - | usage - |
| input sample(s) | input - | input - | input - |
| storage temp. 2-8°C | storage temp. 2-8°C | storage temp. room temp | storage temp. room temp |
| storage condition dry at room temperature | storage condition - | storage condition - | storage condition - |
signalword
Warning
hcodes
Hazard Classifications
Eye Irrit. 2 - Flam. Liq. 3 - Met. Corr. 1 - Skin Irrit. 2
Storage Class
3 - Flammable liquids
flash_point_f
82.4 °F
flash_point_c
28 °C
wgk
WGK 3
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It was stated that free orthophosphate in ATP or GTP is colorimetric. Is it possible to measure CTP using this kit?
1 answer-
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Does your protocol recommend a standard quenching method (e.g., immediate acidification to stop the reaction)? before adding working reagent.
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The working reagent provided in this kit is acidic; therefore, in the case of enzyme reactions, the reaction may be terminated directly by the addition of the working reagent.
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Can EDTA interfere with the formation of the phosphate–molybdate–malachite complex (color development)? Please let me know the acceptable concentration range.
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This kit has not been tested on samples containing EDTA. Based on the chemistry involved one would not expect EDTA to be an interfering substance. However, the suggestion would be to prepare a couple of extra phosphate standards (perhaps the 32, 16 and 8 micromolar standards), and spike them with a solution of EDTA at approximately the same concentration as would be expected in the samples. Make sure that the total volume per well is exactly the same. Compare the un-spiked and spiked standards to see if the added EDTA makes a difference in the absorbance readings for those standards.
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How can I determine the shelf life / expiration / retest date of this product?
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If this product has an expiration or retest date, it will be shown on the Certificate of Analysis (COA, CofA). If there is no retest or expiration date listed on the product's COA, we do not have suitable stability data to determine a shelf life. For these products, the only date on the COA will be the release date; a retest, expiration, or use-by-date will not be displayed.
For all products, we recommend handling per defined conditions as printed in our product literature and website product descriptions. We recommend that products should be routinely inspected by customers to ensure they perform as expected.
For products without retest or expiration dates, our standard warranty of 1 year from the date of shipment is applicable.
For more information, please refer to the Product Dating Information document: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/418/501/product-dating-information-06-25-mk.pdfHelpful?
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Does this kit contain potassium or sodium?
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The standard used in this kit is prepared from potassium phosphate. Sodium is not present in either of the reagents. The specific formulations are considered proprietary.
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Is this kit able to detect pyrophosphate too?
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This kit is intended for the detection of free orthophosphate and has not been tested for pyrophosphate detection. There are other products available that are specifically for use in pyrophosphate detection. Please see the links below to review each item:
MAK168, Pyrophosphate Assay Kit
https://www.sigmaaldrich.com/product/sigma/mak168
MAK169. High Sensitivity Pyrophosphate Assay Kit
https://www.sigmaaldrich.com/product/sigma/mak169Helpful?
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Does the product MAK307 contain magnesium?
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Magnesium is not included as a component in any of the reagents provided with the kit for product MAK307. If magnesium serves as an essential cofactor for the enzyme, it is recommended to supplement it in the buffer before conducting the phosphate determination assay.
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What are the reasons behind the suggestion to use lower concentrations than 0.25mM? Are there potential unwanted biochemical reactions between GTP and malachite green/molybdate if GTP concentration exceeds 0.25mM?
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The kit operates by releasing free orthophosphate from ATP or GTP, followed by the creation of a green complex involving the orthophosphate, molybdate, and Malachite green. It has been observed that samples containing ATP and GTP at concentrations exceeding 0.25mM result in the measurement of free phosphate levels falling outside the detection range of the kit (0.02 µM and 40 µM phosphate), and hence should be avoided.
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Why is it necessary to dilute the ATP concentration to <0.25 mM before the assay? Does ATP interfere with the assay?
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BioAssay Support recommends reducing the ATP concentration before the assay to minimize background interference resulting from non-enzymatic ATP hydrolysis and to decrease the likelihood of reagent precipitation with unhydrolyzed ATP.
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our kit expired, when I used to measure extracted polyphosphate degraded by the enzyme, the results were so different compared to the precious ones (when the kit was not expired), this means the result was not reliable after it expired?
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Correct, products used beyond the expiration date will likely offer poor results.
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