TAKARA MK301 User Manual

Cat. #MK301

TRACP & ALP Assay Kit

v0804

Table of Content

I. Description ................................................................................2

II. Introduction .............................................................................2

III. Kit components .......................................................................2

IV. Storage .......................................................................................3

V. Preparation of reagents .......................................................3

VI. Outline of Procedure ............................................................3

VII. Procedure .................................................................................4

VIII. Application examples ..........................................................5

IX. Reference ............................................................................... 10

X. Related Product ................................................................... 10

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TRACP & ALP Assay Kit

I. Description

TRACP&ALP Assay Kit allows for simultaneous detection of 2 enzymes which are in­volved in bone metabolism. TRACP which is an osteoclast enzyme marker and ALP an
osteoblast enzyme marker. TRACP&ALP Assay Kit has been designed for simple and
quick detection of ACP (Acid phosphatase) and ALP (Alkaline phosphatase) through
the use of pNPP (p-nitro-phenyl phosphate) substrate. The addition of tartaric acid
into the ACP assay, allows for the detection of TRACP (tartrate-resistant acid phos­phatase) activity. Since this kit utilizes an aqueous substrate, it enables quick activity
quantication by measuring the absorbance of the reactant. In addition to this kit,
TRACP & ALP double-staining Kit (Cat.# MK300) is also available using a non-soluble
substrate. The appropriate kit can be selected depending on assay interest.

II. Introduction

Phosphatase is an enzyme which hydrolyzes aliphatic and aromatic phosphate esters
resulting in the release phosphates. The optimum pHs for alkaline and acid phospha­tases activity are at alkaline and acid pHs, respectively. Acid phosphatases (ACP) are
present in a variety of cells and tissues, such as prostate, liver, kidney, spleen, erythro­cyte, platelet and ostelclast.
1,2) In 1959, Burstone3) reported that potent acid phosphatase activity is found in the
osteoclasts and alkaline phosphatase activity is found in the osteoblasts. Following
this report, various research reports have been made on phosphatase activities as­sociated with osteocytes. In addition to osteoclasts, hairy cells among blood cells are
also known to have TRACP activity. The acid phosphatase activity of osteoclasts was
shown to be of the type that retains phosphatase activity in the presence of tartrate
(tartrate-resistant acid phosphatase：TRACP) . The type of acid phosphatases that is
inactivated in the presence of tartrate is called tartrate-sensitive acid phosphatase
(TSACP) . TRACP activity is now a requisite for osteoclasts.
Alkaline phosphatases (ALP) are membrane-bound glycoproteins and are classied
into four types, i.e. intestinal, placental, placenta-like and tissue non-specic types.
Among the tissue non-specic type alkaline phosphatases, the bone-specic isozyme
is called bone type alkaline phosphatase. This enzyme is bound to the membrane of
osteoblasts and functions to enhance osteogenesis by degrading pyrophosphates.
Pyrophosphates, inhibits crystallization at the calcication site and degrads organic
phosphate esters to increase the inorganic phosphate concentration. Therefore, bone
type alkaline phosphatase is known as a marker of osteogenesis in bone cycle me­tabolism.

Cat. #MK301

v0804

Since bone metabolism is composed of mutually balanced osteogenesis and bone
resorption, simultaneous estimation with two enzyme makers is useful.

III. Kit components (for 500 reactions)

1) pNPP (p-nitro-phenyl phosphate) substrate [pNPP substrate] 24 mg x 5 vials
pNPP substrate is supplied su󱐰cient to prepare 25 ml of substrate solution which
allows 500 assays in 50 μ l/well in a 96-well plate.

2) Extraction solution 11 ml x 2 vials.
Physiological saline including 1 % NP-40
For solubilization of suspension and adherent cells

3) Sodium tartrate solution 4 ml

0.5 M sodium tartrate bu󱐯er, pH5.2
TRACP：Used for the detection of osteoclast marker through the addition to the

substrate solution

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TRACP & ALP Assay Kit

4) Bu󱐯er for ACP 30 ml

0.5 M Sodium acetate, pH5.2

5) Bu󱐯er for ALP 30 ml

0.2 M Tris-HCl, pH9.5, 1 mM MgCl
＊ The concentration of Tris-HCl has changed for keeping pH stability from lot. 011.

6) Microplate (96-well) 1 plate
Used in sample dilution or container for reaction
The plate is reusable after soaking in 1 % sodium hypochlorite solution overnight.

Cat. #MK301

v0804

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Reagent required but not supplied in the kit

Stop Solution：0.9 N NaOH
Prior to starting the assay, a customer is required to prepare Stop Solution by himself.
＊：This solution is corrosive. It may cause inflammation when it contacts with

skin. When it comes to contact with hands or mucous membrane, immedi­ately wash away with large amount of water and follow instructions provided
by doctors.

IV. Storage 4 ℃

V. Preparation of Reagents

1) All reagents should be brought to room temperature before use.

2) Preparation of substrate solution

Dissolve 1 vial (24 mg) of [1] pNPP (p-nitro-phenyl phosphate) substrate in 5 ml of
the enzyme bu󱐯er to be assayed ([4] or [5]) , and use this as the substrate solution
(substrate concentration：12.5 mM) . When used for tartrate-resistant acid phos­phatase (TRACP) , sodium tartrate solution [3] should be added at 1/10 the volume
of the substrate solution. In both cases, the prepared reagents should be stored at
－20 ℃ , and used within 1 week.

VI. Outline of Procedure

＜ Cell sample ＞ ＜ Blood sample ＞
Cell washing Serum・Plasma

Cell lysis with Extraction solution dilutio

＊

Selection of Bu󱐯er (for ACP or for ALP)

Enzyme reaction at 37 ℃ for 15 - 60 min.

＜ Endpoint assay ＞
Stop reaction (color formation)

405 nm absorbance

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＜ kinetic assay for ALP only ＞

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