TAKARA MK301 User manual

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
1
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

I. Description
TRACP&ALP Assay Kit allows for simultaneous detection of 2 enzymes which are in-
volved 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 phos-
phatase) 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 phospha-
tases 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, erythro-
cyte, 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 as-
sociated 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 me-
tabolism.
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 Cat. #MK301
v0804
URL：http：//www.takara-bio.com

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 MgCl2
＊ 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.
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, immedi-
ately 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 phos-
phatase (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. ＜ kinetic assay for ALP only ＞
＜ Endpoint assay ＞
Stop reaction (color formation)
405 nm absorbance
3
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

VII. Procedure
(1) For adherent cells cultured in a 96-well plate
1. Remove the culture supernatant with an aspirator, etc.
2. Add 200 μ l of physiological saline to each well, wash once, then discard the liquid.
Note 1) If the cells are detached, this process should be omitted, and a blank well
containing medium only should be set to correct the observed data.
Note 2) Phosphate bu󱐯ers should not be used in washing because they might in-
hibit the enzyme reaction.
3. Pipetting lightly, add 5 - 50 μ l of Extraction solution [2] to each well.
Note 1) The amount of Extraction solution added can be changed depending on
the number of cells. Approximately, 50 μ l should be used in the case of
104 cells.
Note 2) Sample should be diluted with Extraction solution when the sample con-
centration is high. (If extraction solution is insu󱐰cient, physiological saline
can be substituted.)
Part of the diluted sample (5 - 50 μl) should be used in subsequent reactions.
Note 3) Lysis of the cells should be conrmed by microscopic observation.
4. Add 50 μ l of the substrate solution for measurement (see III. Preparation of Re-
agents) to each well and react at 37 ℃ for 15 - 60 minutes.
Note 1) Reaction time can be set arbitrarily.
Note 2) The volume ratio of the cell lysis sample (A) and substrate solution (B)
should be at maximum A：B = 1：1. The cell lysis sample A should be set to
be less than the substrate solution.
Note 3) If high enzyme activity is expected, it is recommended to prepare a di-
luted cell lysis solution for measurement in the provided 96-well plate.
5. Add 50 μ l of stop solution (0.9N NaOH) to each well and measure the absorbance
at 405 nm after color formation.
Note) In the case of acid phosphatase, color formation starts with the addition of
stop solution.
(Reference) Cell lysis sample obtained using the provided Extraction solution [2] can
be used for other measurements besides the target enzyme activity with
this kit, such as protein quantication and kinase activities.
(2) For suspension cells cultured in a Petri dish
1. Collect culture medium with suspension cells into a tube and recover the cells by
centrifugation.
Wash the cells once with physiological saline and precipitate them by centrifuging
again.
2. Add 50 - 500 μ l＊ of Extraction solution [2] to each, and lyse the cells by pipetting.
＊：The amount of extraction solution added can be changed depending on the
number of cells.
Roughly, 50 μ l should be used for 105 cells and 500 μ l for 107 cells.
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TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

3. Dilute the cell lysate step-wise with physiological saline and add the prescribed
amount (in the range of 5 - 50 μ l) to each well of the provided 96-well plate.
Following this step, the procedure is the same as in (1) 4. - 5.
(3) For blood samples
1. Add 5 - 50 μ l of blood samples (serum, plasma) to each well.
Note 1) Sample should be diluted with Extraction solution [2].
Following this step, the procedure is the same as in (1) 4. - 5.
VIII. Application Examples
(1) Measurement of Alkaline Phosphatase (ALP)
This kit was used to measure ALP activity in cultured cells derived from human
small intestine. At the same time, ALP staining was conducted using the xation
solution and insoluble substrate BCIP/NBT included in the TRACP & ALP Double-
stain Kit (Cat. # MK300) .
＜ Method ＞
Suspension of human small intestine cells (Intestine 407) that were serially cul-
tured in 10 % FCS/RPMI1640 medium was added to the rst row of a 96-well plate.
The plate was prepared up to the 11th row by 2-fold dilution (No. of cells：1 x 104
in row 1, 5 x 103 in row 2, and thereafter decreasing by half; row 12 was blank) ,
then further cultured for 2 days (volume：100 μ l/well) . After culturing, ALP stain-
ing was conducted with insoluble substrate BCIP/NBT on columns A and B, and
ALP activity was measured in columns C - F using this kit and Procedure (1) . Due
to solubilization, the volume of extraction solution added to columns C - F varied
from 5 to 50 μ l (5 μ l in column C, 10 μ l in column D, 20 μ l in column E, 50 μ l
in column F) . Enzymes reactions were conducted for 60 minutes at 37 ℃ .
＜ Results ＞
Alkaline phosphatase activity is extremely strong in the small intestine cells, and
the correlation of activities and number of cells were conrmed using this kit.
The detection sensitivity for human small intestine cells (Intestine 407) under
the conditions of this activity measurement was 10 cells/assay. It was conrmed
that even though the volume of extraction solution added varied in the range of
5 to 50 μ l/well, there was little inuence on the enzyme reactions. There is no
problem in varying the volume of extraction solution within a xed range if the
cells are solubilized at a su󱐰cient volume.
5
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

The staining of ALP was carried out as follows.
1 x 104 cells / well
(2) Measurement of Tartrate-resistant Acid Phosphatase (TRACP)
＜ Method ＞
Bone marrow cells derived from the femur of 16-week-old rabbit were suspended
in 10 % FCS/RPMI1640 medium. This cell suspension was added to the rst row of
a 96-well plate, and the plate was prepared up to the 11th row by 2-fold dilution
of the same culture (No. of cells：7 x 105 in row 1, 3.5 x 105 in row 2, and thereafter
decreasing by half in the same manner; row 12 was blank) , at a volume of 100 μ l/
well, and culturing was initiated. A further 100 μ l of medium was added because
adhesive cells had become prominent after 3 days, and culturing was continued.
Eight days after seeding, TRACP activity was measured using the kit according to
procedure (1) .
The degree of substrate color formation was compared when substrate solution
was directly added to the adhesive cells without extraction procedure and when
they were solubilated with 25 μ l of extraction solution. 50 μ l of substrate solution
for TRACP was used, and enzyme reactions were conducted for 60 minutes at 37 ℃ .
Plate row 1 2 3 4 5 6
No. of cells
(cells/well) 1 × 1045 × 1032.5 × 1031.25 × 1036.25 × 1023.13 × 102
A405 Plate Column
C 5 μ l ＊2.275 2.271 1.732 0.920 0.581 0.329
D 10 μ l ＊2.298 2.263 1.957 1.082 0.719 0.373
E 20 μ l ＊2.289 2.273 1.901 1.101 0.617 0.369
F 50 μ l ＊2.236 2.214 1.964 1.287 0.744 0.341
＊：Volume of extraction solution added
7 8 9 10 11 12
1.56 × 1027.8 × 1013.9 × 10220 10 0
0.239 0.180 0.160 0.158 0.154 0.145
0.252 0.200 0.176 0.154 0.158 0.148
0.228 0.192 0.168 0.162 0.152 0.145
0.234 0.175 0.161 0.152 0.157 0.145
6
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

＜ Results ＞
The bone marrow cell culture produces numbers of naturally di󱐯erentiated osteo-
clast-like TRACP-positive cells, and their TRACP activity could be measured using
this kit. The table below shows the numbers of cells at the start of culturing and
TRACP activity as absorbency at 405 nm. Similar levels of activity were detected in
the live cells to which substrate was directly added without extraction (nal sub-
strate concentration 12.5 mM) and in cells on which extraction was conducted (nal
substrate concentration 4.1 mM) , and results that correlated with the numbers of
cells were obtained.
No. of cells (cells/well) 7 × 1053.5 × 1051.75 × 1058.76 × 1044.38 × 1042.19 × 104
Substrate directly added 1.701 1.471 0.747 0.295 0.156 0.120
Extracted 1.754 1.880 0.781 0.246 0.150 0.125
1.1 × 1035.5 × 1022.25 × 102112 56 0
0.105 0.100 0.104 0.102 0.100 0.100
0.112 0.111 0.112 0.107 0.105 0.108
(3) Measurement with Acid Phosphatase (ACP) Standards
A standard curve was made using ACP (Code 108227, Lot 93207721) from Roche
Diagnostics.
Distilled water was added to the reference standards to prepare 100 μ g/ml en-
zyme solutions, and a 2-fold dilution series was prepared using each enzyme buf-
fer.
Using 1 well of a 96-well plate for 1 reaction, 50 μ l of enzyme and 50 μ l of the
substrate solution ＊ were mixed and reacted for 30 minutes at 37 ℃ . After adding
50 μ l of stop solution, absorbency at 405 nm was immediately measured using a
plate reader.
Note) In the case of acid phosphatase, color forms with the addition of stop solution.
＊：Tartaric acid is not added to acid phosphatase substrate.
＜ Results ＞
In the reaction scales produced：
For ACP, concentrations were measurable from 1.5 μ g/ml or less, and actual lin-
earity was obtained in the range up to 0.5 μ g/ml.
7
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

Acid phosphatase 100 μ l/ml preparation：
21 ～ 215 stepwise dilution
μ l/ml 405 nm
50 4.014
25 3.993
12.5 3.995
6.26 4.066
3.125 4.011
1.5625 4.032
0.78125 3.411
0.390625 2.043
0.195313 1.147
0.097656 0.599
0.048828 0.356
0.024414 0.215
0.012207 0.157
0.006104 0.120
0.003052 0.108
0 0.084
(4) Measurement with Alkaline Phosphatase (ALP) as standard sample
A standard curve was made using 3 types of alkaline phosphatase, BAP (Cat.#
2120A, Lot K2601EA), CIAP (Cat. # 2250A, Lot E2301AB), or SAP (Cat.# 2660A, Lot
N301CB) of TAKARA BIO INC.
The 2-fold dilution series of the enzyme solutions, which be added Bu󱐯er for ALP
in the kit to each enzyme, was prepared with Extraction solution in the kit. Both
50 μ l of enzyme solution and 50 μ l of pNPP substrate dissolved in the Bu󱐯er for
ALP were added and mixed in one well of 96-well plate, and incubated at 37 ℃ for
30 minutes. Immediately after stopped by adding 50 μ l of Stop solution, absor-
bance at 405 nm of the 96-well plate was measured using a plate reader.
＜ Result ＞
All alkaline phosphatase (BAP, CIAP, and SAP) from TAKARA BIO were available as a
positive control for the kit.
(5) Comparison of Tartrate-resistant Acid Phosphate (TRACP) and Alkaline Phospha-
tase (ALP) by he preparation method of blood samples.
＜ Method ＞
Blood was collected from three rabbits simultaneously and the collocted blood
samples were preared by three methods (citrate plasma (PPP) , serum and hemoly-
sis serum) . 50 μ l of serial dilution samples and 50 μ l of the respective substrate
solution were mixed and reacted for 30 minutes at 37 ℃ . After adding 50 μ l of
stop solution, absorbance at 405 nm was measured using a plate reader. All of the
samples were measured simultaneously on the day when blood samples were
prepared.
Acid phosphatase standard
μ g/ml
8
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

＜ Results ＞
＜ TRACP activity ＞ A405 nm absorbanse
TRACP citrate plasma（ PPP ） serum hemolysis serum
ID No. x 20 x 40 x 80 x 20 x 40 x 80 x 20 x 40 x 80
Rb No. 1 0.821 0.420 0.247 0.834 0.487 0.309 0.834 0.487 0.300
Rb No. 2 1.045 0.520 0.311 0.704 0.422 0.268 1.066 0.582 0.360
Rb No. 3 0.702 0.370 0.237 1.000 0.579 0.353 0.768 0.360 0.275
＜ ALP activity ＞ A405 nm absorbanse
ALP citrate plasma（ PPP ） serum hemolysis serum
ID No. x 2 x 4 x 8 x 2 x 4 x 8 x 2 x 4 x 8
Rb No. 1 0.514 0.321 0.217 0.801 0.469 0.295 1.074 0.599 0.374
Rb No. 2 0.481 0.299 0.206 0.528 0.327 0.217 0.801 0.483 0.304
Rb No. 3 0.348 0.231 0.165 0.718 0.431 0.275 0.650 0.398 0.252
As the phosphate activity is di󱐯erent depending on the preparation method of
blood sample, it is necessary to use the sample of the same preparation method.
(6) Inuence of freeze-thaw cycles of blood samples on the phosphate activity.
＜ Method ＞
Serum samples were collected from three rabbits. Samples were devided into four
portion and each sample was repeated various cycles of freeze-thawing ( － 80 ℃
⇔ 25 ℃）
For the measurement of TRACP (tartrate-resistant acid phosphate), samples were
diluted by 20-, 40- and 80- fold. For the measurement of ALP (alkaline phosphate),
samples were diluted by 2-, 4- and 8- fold.
50 μ l of serial dilution samples and 50 μ l of the respective substrate solution
were mixed and reacted for 30 minutes at 37 ℃ . After adding 50 μ l of stop solu-
tion, absorbance at 405 nm was measured using a plate reader. All of the samples
were measured simultaneously on the day when serum samples were prepared.
＜ Results ＞
＜ TRACP activity ＞ A405 nm absorbanse
TRACP no freezing freezing-thaw 1 cycle freezing-thaw 2 cycles freezing-thaw 3 cycles
ID No. ｘ 20 ｘ 40 ｘ 80 ｘ 20 ｘ 40 ｘ 80 ｘ 20 ｘ 40 ｘ 80 ｘ 20 ｘ 40 ｘ 80
Rb No. 1 0.826 0.477 0.295 0.832 0.438 0.284 0.778 0.452 0.292 0.736 0.432 0.277
Rb No. 2 1.087 0.577 0.342 1.078 0.573 0.343 1.021 0.580 0.353 0.980 0.565 0.344
Rb No. 3 0.743 0.409 0.252 0.754 0.429 0.269 0.749 0.434 0.279 0.710 0.424 0.275
＜ ALP activity ＞ A405 nm absorbanse
ALP no freezing freezing-thaw 1 cycle freezing-thaw 2 cycles freezing-thaw 3 cycles
ID No. ｘ 2 ｘ 4 ｘ 8 ｘ 2 ｘ 4 ｘ 8 ｘ 2 ｘ 4 ｘ 8 ｘ 2 ｘ 4 ｘ 8
Rb No. 1 0.616 0.338 0.281 0.602 0.328 0.212 0.592 0.335 0.214 0.588 0.330 0.221
Rb No. 2 0.577 0.327 0.206 0.600 0.338 0.214 0.562 0.324 0.209 0.577 0.334 0.213
Rb No. 3 0.400 0.243 0.164 0.423 0.255 0.170 0.440 0.261 0.175 0.415 0.248 0.173
It seemed that both of TRACP and ALP activities were not comparatively influ-
enced by freeze-thaw cycles of blood samples, but it was preferable even twice of
freeze-thaw cycles in the same condition.
9
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com

IX. Reference
1) Burstone, M. S. et al . (1958) J. Natl. Cancer Inst. 20, 601-615.
2) Burstone, M. S. et al. (1958) J. Natl. Cancer Inst. 21, 523-539.
3) Burstone, M. S. (1959) J. Histochem. Cytochem . 7, 39-41.
4) Harlow and Lane (1988) Antibodies, A LABORATORY MANUAL, 406- 407.
X. Retated Products
TRACP & ALP double-stain Kit (Cat. # MK300)
Note： This product is intended to be used for research purpose only. They are not to be used for
drug or diagnostic purposes, nor are they intended for human use. They shall not to be used
products as food, cosmetics, or utensils, etc.
Takara products may not be resold or transfered, modied for resale or transfer, or used to
manufacture commercial products without written approval from TAKARA BIO INC.
If you require licenses for other use, please call at [phone] or contact from our web-
site at www.takara-bio.com .
Phone：[phone] Fax：[phone]
10
TRACP & ALP Assay Kit Cat. #MK301
v0804
URL：http：//www.takara-bio.com