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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

Summary of Contents for MK301

Page 1: ...duction 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 TRACP ALP Assay Kit Cat MK301 v0804 URL http www takara bio com ...

Page 2: ...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 boun...

Page 3: ...ded 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 buffer 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 s...

Page 4: ...s 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 so...

Page 5: ... 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 col...

Page 6: ... 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 we...

Page 7: ...102 112 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...

Page 8: ...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...

Page 9: ... 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 freezi...

Page 10: ...ly 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 modified 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 81 77 543 7247 ...

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