Wako 残留DNA提取试剂盒 DNA with DNA Extractor® Kit

Wako 残留DNA提取试剂盒 DNA with DNA Extractor® Kit

This article was written by Hiroki Fukuchi, Life Science Research Laboratories of FUJIFILM Wako Pure Chemical Corporation, Japan.

导言
因为许多生物药物,包括疫苗,都是由培养的细胞或微生物(如大肠杆菌指出宿主细胞来源的DNA可能存在于合成的药物物质和产物中.鉴于残留DNA可能转移宿主细胞或病毒衍生的癌基因,病毒DNA可能导致感染事件,定量检测残留DNA是制造业的重要测试,也是生物制药过程验证等测试的一部分。正如一份报告所指出的那样,每一剂量的残余DNA最多可达100 pg,这对于生物制药来说是可以接受的,1)不仅在美国和欧洲国家,而且在其他国家,越来越需要对宿主细胞中的残留DNA进行定量检测。为了检测残留DNA的痕迹,必须从样品中提取出高收率的残留DNA。本文旨在介绍DNA提取器的用途。®用于检测残留DNA作为DNA纯化试剂的试剂盒。

DNA提取器®试剂盒(#295-50201,Fujifilm Wako纯化学公司)
在对生物制品中的总DNA进行检测和定量之前,有必要从蛋白质等其他生物成分中分离纯化生物制品中的DNA。一般来说,DNA的分离是通过蛋白酶介导的样品消化,然后用苯酚和/或氯仿提取的。然而,这种方法的缺点是,必须使用有害的苯酚和氯仿,需要时间和人力的提取,虽然可以获得相当高纯度的DNA。此外,二氧化硅载体固相萃取,由于载体吸附导致DNA丢失,对DNA的提取效果不理想。有机溶剂的提取也不利于DNA的有效回收,这是DNA提取试剂需要解决的关键问题之一。

DNA提取器®试剂盒(产品代码:295-50201),我们于1992年推出,解决了上述限制,使用钠碘提取高纯度的DNA,通过简单的程序。

DNA提取原理®试剂盒
工具包的组成部分*含有碘化钠和表面活性剂,当2-丙醇加入时,它们通过溶解蛋白质和其他生物成分,选择性地沉淀(共沉淀)核酸(主要是DNA)和糖原,作为蛋白质增溶剂(潮致离子)。2)与上述方法不同,纯化步骤被简化为在没有载体或有机溶剂的情况下产生沉淀物,从而能够以较高的收率提取微量DNA。

*工具包的组成部分:
碘化钠溶液,N-月桂酸盐溶液,洗涤液(A),
洗涤液(B),糖原溶液

DNA提取器提取DNA的实例®试剂盒和总DNA定量
本文介绍了一份关于在稀释剂或辅料存在的情况下DNA产量的报告,该报告最初发表在瓦科纯化学杂志第60卷第3期(1992年)第28页。本实验通过在磷酸盐缓冲液中溶解常用作稀释剂或辅料(如精氨酸、尿素)或蛋白质(BSA和人γ-球蛋白)的常用或过量的物质制成模型溶液,并在每个模型溶液中加入一定量的小牛胸腺脱氧核糖核酸(PG)。然后,根据dna提取器标签上的说明,从模型溶液的400μL中提取dna。®基特。所得沉淀物溶于500μL磷酸盐缓冲液中,用阈值法测定总dna。®总DNA分析系统,*3,4)来测量DNA的产量。测量结果见表1。

Introduction

Since many biopharmaceuticals, including vaccines, are manufactured from cultured cells or microorganisms such as Escherichia coli, it is pointed out that host cell-derived DNA may remain in resultant drug substances and products. Given the possibility that the residual DNA may transfer host cell- or virus-derived oncogenes and that viral DNA may cause infectious events, quantitative detection of residual DNA is important testing for manufacturing and as part of testing such as process validation for biopharmaceuticals. As indicated by a report recommending that up to 100 pg of residual DNA per dose is acceptable for biopharmaceuticals,1) there is increasing need for quantitative detection of residual DNA from host cells as a quality test not only in the US and European countries but also in other countries. To detect traces of residual DNA, it is necessary to extract traces of residual DNA from a sample at a high yield. This article is intended to introduce the usefulness of DNA Extractor® Kit in detecting traces of residual DNA as a DNA purification reagent.

DNA Extractor® Kit (#295-50201, FUJIFILM Wako Pure Chemical Corporation)

Prior to detection and quantification of total DNA in a biologics, it is necessary to separate and purify DNA in the biologics from other biological components such as proteins. In general, DNA is isolated through protease-mediated digestion of specimen followed by extraction with phenol and/or chloroform. However, this method has disadvantages that deleterious phenol and chloroform have to be used and that time- and labor-consuming extraction is required, although quite highly pure DNA can be obtained. In addition, solid-phase extraction via silica carriers, which results in loss of DNA due to adsorption on carriers, is not ideal to recover traces of DNA. Extraction with organic solvents is also not favorable with inefficient recovery of traces of DNA, which is a key issue to be addressed with DNA extraction reagents.

DNA Extractor® Kit (Product code: 295-50201), which we launched in 1992, resolves the aforementioned limitations by using sodium iodide to extract highly pure DNA at high yields through simple procedures.

Principle of DNA Extractor® Kit

The components of the kit* contain sodium iodide and surfactants, which act as protein solubilizers (chaotropic ions) by solubilizing proteins and other biological components in specimens to precipitate (coprecipitate) nucleic acids (mainly DNA) and glycogen selectively when 2-propanol is added.2) Purification steps are simplified to produce precipitates without carriers or organic solvents, unlike the aforementioned methods, enabling the extraction of traces of DNA at high yields.

* Components of the kit:
Sodium Iodide Solution, Sodium N-Lauryl Sarcosinate Solution, Washing Solution (A),
Washing Solution (B), Glycogen Solution

Example of DNA extraction with DNA Extractor® Kit and total DNA quantification

A report on yields of DNA in the presence of diluents or excipients with the use of the kit, which was originally published on page 28 in Wako Pure Chemical Jiho Vol. 60 No. 3 (1992), is introduced here. In this experiment, model solutions were prepared by dissolving usual or excessive doses of substances often used as diluents or excipients (e.g., arginine, urea) or proteins (BSA and human γ-globulin) in phosphate-buffered saline, and a given amount (pg) of calf thymus DNA was added to each model solution. Subsequently, DNA was extracted from 400 μL of model solution according to the instructions on the label of DNA Extractor® Kit. The obtained precipitates were dissolved in 500 μL of phosphate-buffered saline, and total DNA was quantified using Threshold® Total DNA assay system,*3,4) to measure the yield of DNA. Measurement results are presented in Table 1.

Table 1. Yields of DNA in the presence of diluents or excipients
Diluent and its amount Amount of DNA (pg) Yield of DNA (%)
Sorbitol 200 mg/mL
50 mg/mL
50
50
95
94
Maltose 400 mg/mL
200 mg/mL
50 mg/mL
50
50
50
89
102
98
Mannitol 200 mg/mL 50 84
Dextrose 200 mg/mL 50 81
Sucrose 200 mg/mL 50 92
Urea 1 mol/L 50 106
Arginine 200 mg/mL 50 110
γ-globulin 60 mg/mL
60 mg/mL
60 mg/mL
10
5
2.5
102
84
88
BSA 200 mg/mL 10 95

For all five sugars at one or more concentrations, 80% to 110% of DNA was recovered. For arginine and urea, DNA was extracted at a high yield. For both BSA and human γ-globulin, the proteins tested, the yield of DNA was high (some proteins in specimens may precipitate, but the precipitation can be managed with dilution or proteolytic enzymes5)). The aforementioned results show that the kit can extract residual DNA from various samples at high yields in a highly reproducible manner.

* Threshold® Total DNA assay system is manufactured by Molecular Devices for total DNA quantification and designed to quantitatively measure DNA as a molecule rather than as a gene. This system has a detection sensitivity of 2 pg/assay for total DNA.

Example of extraction of traces of residual DNA with DNA Extractor® Kit and DNA quantification by qPCR assay

As mentioned above, Threshold® Total DNA assay system is intended to quantify total DNA, but not to detect any specific gene. Considering the advantages of qPCR quantification for detecting a specific gene, which can be used to detect residual DNA clearly derived from host cells more sensitively than total DNA quantification with Threshold® Total DNA assay system, it was investigated whether traces of DNA could be extracted from potential host cells.

In this experiment, genomic DNA from CHO cells or Escherichia coli, which are widely used to produce proteins and antibodies, was used as a residual DNA model to extract and quantify traces of residual DNA using a qPCR assay. The experimental procedure is presented in Figure 1.

Figure 1. Experimental procedure

wb021698_img01.png

First, DNA was extracted from samples spiked with 0.1 to 100 pg of genomic DNA in 100 μL of water (distilled water for injection) using the kit and then dissolved in 100 μL of water. Subsequently, a qPCR assay was performed to calculate the yield of DNA from a calibration curve prepared simultaneously. The yield of DNA was 85% to 120% for Escherichia coligenome in the range of 1 to 100 pg and for CHO cell genome in the range of 0.1 to 100 pg (the yield was almost 100% up to 1,000 pg for both genomes, although no data are presented here).

Another experiment was performed based on the assumption that residual DNA was contained in a cell culture supernatant. After Panc-1, a human pancreatic cancer-derived cell line, was cultured in 10% FBS DMEM for 3 days, 0.1 pg of genomic DNA from CHO cells was added to 500 μL of culture supernatant to prepare a cell culture supernatant sample. The yield was calculated to be 0.093 pg from a calibration curve. This experiment showed that 0.1 pg (100 fg) of residual DNA in a 500-μL sample, as small as trace in fg units, can be extracted at a high yield using the kit. In addition, traces of residual DNA was recovered from all samples, including water, phosphate-buffered saline, and cell culture supernatant, at as high yields as almost 90% or more (Tables 1 and 2), suggesting that the kit can extract traces of residual DNA from various samples at high yields, as mentioned in the section of total DNA quantification.

Table 2. Yields of genomic DNA
Sample
(added genomic DNA)
Amount of DNA Amount of recovered DNA determined from calibration curve Yield
Distilled water for injection
(Escherichia coli)
0.1pg ND (less than lower limit of detection)
1pg 1.031pg 103%
10pg 11.09pg 111%
100pg 85.1pg 85%
Distilled water for injection
(CHO cells)
0.1pg 0.933pg 93%
1pg 1.187pg 119%
10pg 11.57pg 116%
100pg 87.1pg 87%
Human cell culture supernatant (CHO cells) 0.1pg 0.0928pg 93%

Conclusion

Residual DNA was recovered from samples at high yields using the kit. DNA extraction from samples is a very important step for quantifying residual DNA, and the kit can extract traces of residual DNA at high yields. In addition, since the kit can be used in various samples and may be useful in extracting residual DNA not only from CHO cells but also from other host cells such as Escherichia coli and yeast, the kit is expected to be used for testing of biopharmaceuticals in future.

References  参考文献

  1. Knezevic et al, Biologicals 36:203-211 (2008)
  2. Ishizawa, M et al, Nucleic Acids Res., 19, 5792 (1991)
  3. Kung. V. T et al, Anal. Biochem. 187, 220-227 (1990)
  4. Mizusawa S, Honma R, Pharm Tech. Japan 7, 309-314, 426-431 (1991)
  5. Wako Pure Chemicals Jiho Vol. 61 No. 1 p. 27 (1993)

1.Related product
DNA Extractor® KitRelated Products
DNA Extractor® Kit Series

Wako 和光纯药 011-27991 Anti Iba1, Goat Iba1抗体,山羊源多克隆抗体说明书

Wako 和光纯药 011-27991 Anti Iba1, Goat  Iba1抗体,山羊源多克隆抗体说明书

【Background】
Iba1 is a protein highly expressed in microglia and macrophages with a molecular weight of about 16.7 kDa1). The protein is a commonly known microglial marker in the nervous system.This item is a goat polyclonal antibody that reacts with Iba1.
For Research Use Only. Not for use in diagnostic procedures or therapeutic use.

小胶质细胞标记
Iba1是一种约17 kDa的蛋白,在神经系统小胶质细胞中特异性表达,经常被用作小胶质细胞标记物。 本产品是识别Iba1的山羊多克隆抗体。

【Description】

[Purification] Purified from the goat serum by antigen-affinity chromatography
[Reactivity] Reacts with Iba1
[Antigen] Synthetic peptide corresponding to the C-terminus of Iba1
[Clone No.] -( polyclonal)
[Species cross reactivity] Mouse and Rat(Other species have not been tested)
[Host] Goat
[Concentration] indicated on the label
[Formulation] TBS

[纯化]通过抗原亲和层析从山羊血清中纯化
[反应性]与Iba1反应
[抗原]对应于Iba1 C末端的合成肽
[克隆号]-(多克隆)
[物种交叉反应]小鼠和大鼠(尚未测试其他物种)
[主持人]山羊
标签上指示的[浓度]
[配方] TBS

【Applications】
Immunohistochemistry( frozen section) 1 : 250-1,000
Immunohistochemistry( paraffin section) 1 : 250-1,000
Western Blot 1 : 1,000
Optimal concentration should be determined by each laboratory
for each application.
【Storage】
Store at -20℃ .  Avoid repeated freeze and thaw.
【Package】
100μL
【Recommended protocol( Immunohistochemistry frozen section)】
Wistar rat or ICR mouse was perfusion-fixed with 4% paraformaldehyde.
replaced sucrose, and prepared 25μm brain section
by microtome.
Wash : 0.3% TritonX-100 in PBS, 5 min × 3

Blocking : 1% BSA and 0.3% TritonX-100 in PBS, 2 hour, RT

Primary antibody : goat anti-Iba1 (1/1000), 1% BSA, and 0.3%

TritonX-100 in PBS, overnight, 4℃

Wash : 0.3% TritonX-100 in PBS, 5 min × 3

Secondary antibody : AlexaFluor488 anti-goat IgG (1/1000,
Jackson Immuno Research Laboratories #705-545-147), 1%
BSA, and 0.3% TritonX-100 in PBS, 2 hour, RT

Wash : 0.3% TritonX-100 in PBS, 5 min × 3

Mount
【Reference】 参考文献
1) Imai, Y., Ibata, I., Ito, D., Ohsawa, K. and Kohsaka, S. : Biochem.
Biophys. Res. Commun., 224, 855( 1996).

◆应用实例 1:免疫组织染色(荧光染色)

Immunohistochemistry (fluorescent staining)

03700482_img04.png

数据提供:国立长寿医疗研究中心榊原老师

样品:阿尔茨海默病模型小鼠(APPNL-G-F 小鼠)大脑新皮层冰冻切片

一抗:抗Iba1,山羊多克隆抗体(1:1,000)

二抗:Alexa Fluor488标记抗山羊IgG

Aβ染色:0.001 % FSB溶液(淀粉样蛋白染色荧光探针)

数据提供:创价大学理工学部中嶋老师

样本:大鼠(左)以及小鼠(右)大脑皮质冰冻切片

一抗:抗Iba1,山羊多克隆抗体(1:250)

二抗:Alexa Fluor488标记抗山羊IgG

◆应用实例 2:免疫组织染色(DAB染色)

03700482_omg02.png

样品:小鼠脑额叶石蜡切片

一抗:抗Iba1,山羊(1:1,000)

二抗:抗山羊IgG,生物素标记

抗原激活:10 mM柠檬酸盐缓冲液(pH 6),90°C,处理10 min

◆应用实例3:蛋白印迹 Western Blotting

03700482_omg03.png

数据提供:创价大学理工学部中嶋老师

样品:大鼠原代培养小胶质细胞  10 μg

样品:大鼠原代培养神经元 10 μg

样品:大鼠原代培养星形胶质细胞  10 μg

样品:大鼠大脑皮层  100 μg

一抗:抗Iba1,山羊(1:1,000)

二抗:抗山羊IgG,HRP标记

抗原 合成肽(Iba1 C端序列相同)
储存缓冲液 TBS
亚型 山羊IgG
物种交叉性 大鼠、小鼠
抗体浓度 0.5-0.6 mg/mL
应用 免疫组化(冰冻切片)1:250-1,000
免疫组化(石蜡切片)1:250-1,000
免疫印迹 1:1,000

◆产品信息

产品编号 产品名称 规格 包装
011-27991 Anti Iba1, Goat

抗Iba1,山羊源多克隆抗体

免疫化学用 100 μL

◆相关产品

产品编号 产品名称 规格 包装
019-19741 Anti Iba1, Rabbit (for Immunocytochemistry)
小胶质细胞/巨噬细胞特异性蛋白抗体(免疫组化)
免疫化学用 50 μg
013-27691 Anti Iba1, Rabbit(for Paraffin Section)
小胶质细胞/巨噬细胞特异性蛋白兔源抗体(石蜡切片)
免疫化学用 50 μg
016-26461 Anti Iba1, Rabbit, Biotin-conjugated
小胶质细胞/巨噬细胞特异性蛋白抗体(结合生物素)
免疫化学用 100 μL
013-26471 Anti Iba1, Rabbit, Red Fluorochrome(635)-conjugated
小胶质细胞/巨噬细胞特异性蛋白抗体(结合红色荧光素635)
免疫化学用 100 μL
016-20001 Anti Iba1,Rabbit (for Western Blotting)
小胶质细胞/巨噬细胞特异性蛋白抗体(免疫印迹)
免疫化学用 50 μg
012-26723 Anti Iba1, Monoclonal Antibody(NCNP24)
抗Iba1,单克隆抗体(NCNP24)(鼠源)
免疫化学用 10 μL
017-27591 Anti Human Iba1, Monoclonal Antibody(NCNP27)
抗人 Iba1,单抗(NCNP27)
免疫化学用 10 μL

WAKO 295-50201 DNA Extractor Kit 生物制药残余DNA抽提试剂盒

WAKO 295-50201 DNA Extractor Kit  生物制药残余DNA抽提试剂盒

DNA Extractor® Kit

for Genetic Research
Manufacturer :
FUJIFILM Wako Pure Chemical Corporation
Storage Condition :
Keep at 2-10 degrees C.
产品描述

血清、血浆可用。(更新日期:20190621)

传统的方式是采用蛋白酶-SDS的方法提取DNA,虽然能够获得高品质的DNA,但是后续需要用酚-氯仿进行处理,而酚-氯仿属于危险化学品,对人体有一定危害。

该试剂盒采用碘化钠法,能获得高质量和高回收率的DNA,抽提过程不需要酚-氯仿。

碘化钠代替酚-氯仿,溶解蛋白和脂类。糖原作为载体,异丙醇沉淀DNA。

只需将试剂加入离心管内即可,不像Qiagen的DNA抽提试剂盒采用柱吸附法。

产品回收率10pg左右。

文献报道的回收率是80%~90%。

Kit component

Kit component
50 tests
Sodium Iodide Solution 2 x 26 mL
Sodium N-Lauryl Sarcosinate Solution 1 x 1.2 mL
Washing Solution (A) 1 x 42 mL
Washing Solution (B) 2 x 40 mL
Glycogen Solution 1 x 0.1 mL

Features

  • Efficient recovery of trace DNA (100-1,000 fg)
  • Perform in a single tube for complete the process
  • Extraction completed in 60-90 minutes
  • Pretreatment protocol for samples containing high protein concentration
  • Adopts Sodium Iodide method*

*Sodium Iodide method is a residual DNA extraction technique that is described in The United States Pharmacopeia (USP) 42-NF37, <509> Residual DNA Testing.

Principle

Principle of DNA extraction

i. Cell membrane and cytoplasm are destroyed by surfactant, isolating cell nuclei.
ii. The nuclear membrane and nuclear protein are decomposed by protease to expose DNA.
iii. Protein and lipids are made soluble by the action of sodium iodide, and DNA is precipitated with isopropanol.

Extraction of viral DNA in serum and an infinitesimal amount of fungus-derived DNA in biological products

 

  • Extraction from serum

    01413_img01.png

    Operation time: 1 to 1.5 hours
    Sample amount: 100 μL/test

  • This kit is intended for use in extracting host cell-derived residual DNA remaining in biological components using the Sodium Iodide method. Extracted DNA can be quantified by qPCR. Use the DNA Extractor® kit for testing and management of the amount of DNA derived from host cells such as CHO cells, Escherichia coli, and yeast. DNA from any species extracted using this kit is suitable for using the Threshold Immunoassay Syste® provided by Molecular Devices, LLC. The kit can also be used for DNA extraction from serum.

Data

Recovery test of CHO cell-derived DNA

Purpose:
To examine the yields of CHO cell-derived DNA from culture supernatant.

Methods:
Using the kit, DNA was extracted from culture supernatants of PANC-1 cells to which 10 fg to 1 ng of CHO cell-derived DNA was added. A qPCR assay was then performed using the extracted DNA to obtain Cq values.
A qPCR was also performed using purified water to which CHO cell-derived DNA was added, without DNA extraction (standard conditions), to obtain Cq values. The DNA yield under each set of conditions was calculated based on a calibration curve generated from the results of the standard conditions.

Samples:
1. Purified water containing CHO cell-derived DNA (no DNA extraction): Standard conditions
2. DNA extracted from the culture supernatants of PANC-1 cells containing CHO cell-derived DNA, using the kit.

qPCR reagents:
・GeneAce SYBR® qPCR Mix α No ROX (Nippon Gene#319-07703)
・Optical Flat 8–Cap Strips for 0.2 mL tube strips/Plates (BIO-RAD#TCS0803)
・Hard-Shell® Thin-wall 96-well PCR plates (BIO-RAD#HSP9601)

Results:

1. Standard conditions 2. Culture supernatants
Added DNA (fg) Cq 1 Cq 2 Mean Cq 1 Cq 2 Mean
0 ND
10
100 36.89 ND 36.89 37.44 36.73 37.09
1,000 33.44 33.91 33.68 34.09 34.01
10,000 30.23 30.17 30.20 30.72 30.96 30.84
100,000 26.74 26.68 26.71 26.90 27.03 26.96
1,000,000 23.39 23.28 23.33 23.61 23.48 23.54

ND: Not detected

01413_img20.png

DNA added to culture supernatants (100 fg to 1 ng) was extracted with high yield.

This test was performed in accordance with Protocol #2 described in the manual.

Recovery test of E. coli-derived DNA

Purpose:
To examine the yields of E. coli-derived DNA.

Methods:
E. coli-derived DNA (10 fg to 1 ng) was added to purified water, and Cq values were measured before and after DNA extraction using the kit. Calibration curve was generated from mean Cq values of samples before the DNA extraction to calculate the yields after the extraction.

Samples:
Purified water containing E. coli-derived DNA

qPCR reagents:
・GeneAce SYBR® qPCR Mix α No ROX (Nippon Gene#319-07703)
・Optical Flat 8–Cap Strips for 0.2 mL tube strips/Plates (BIO-RAD#TCS0803)
・Hard-Shell® thin-wall 96-well PCR plates (BIO-RAD#HSP9601)

Results:

01413_img21.png

E. coli-derived DNA added to the samples (1 pg to 1 ng) was extracted with high yield.

This test was performed in accordance with Protocol #1 described in the manual.

Examples of using DNA Extractor® Kit

01413_img02.png

Reference

Ishizawa, M. et al., Nucleic Acids Res., 19, 5792 (1991).

Overview / Applications

Outline This is for research use only. Do not administer it to human. Molecular Biology-DNA Extraction Kits. Detects and quantitates contaminant DNA in serum and residual DNA in biopharmaceuticals. Employs a new extraction procedure for DNA purification from a single tube. This procedure, using Sodium Iodide (NaI) as a chaotropic agent, realizes DNA isolation of both high quality and high recovery from biological fluids without the use of phenol or chloroform. Complex and laborious manipulations are avoided when using this kit. In addition, this kit has a modified application for use with Molecular Devices’ Threshold(TM) System.
PRINCIPLE of DNA EXTRACTION: A high concentration of chaotropic reagent, NaI, and an anionic detergent participate in solubilization of the proteins and lipids contained in biological samples. After addition of isopropanol to the mixture, nucleic acids are co-precipitated with polysaccharide glycogen as a carrier, while other components remain soluble in the solution phase.Ref.: Ishizawa, M., Kobayashi, T. and Matsuura, S., ”Simple procedure of DNA Isolation from Human Serum”, Nucleic Acids Res.19, 5792 (1991).This method was listed on USP39-NF34, page 1413/Pharmacopeial Forum. Vol.41(4).
Purpose DNA extraction.

Related Information  其他相关产品:
Similar items list
DNA Extractor® Kit Series
Product List
Product Name
DNA Extractor® WB Kit (Sodium Iodide method) for Whole Blood DNA Extraction
DNA Extractor® SP kit for Genetic Research
DNA Extractor® TIS Kit for Genetic Research
DNA Extractor® FM Kit
8-OHdG Assay Preparation Reagent Set for Genetic Research

Wako 分散酶 Dispase CAS 9001-92-7

Wako 分散酶 Dispase CAS 9001-92-7

Dispase分散酶

Dispase分散酶是一类中性金属蛋白水解酶,可以回收生长在Matrigel基质上的细胞。与胰酶、胶原酶相比,它的作用更加温和,所以不会损伤细胞。此外,分散酶也可以用于组织分离。

来源:多粘芽孢杆菌表达的金属蛋白酶
使用指南:推荐浓度为10 U/cm2 BD Matrigel基质,如35 mm的培养皿推荐使用浓度为100U。
保存条件和效期:-20℃下冷冻保存,避免多次冻融。保质期详见产品包装。

Wako 分散酶 Dispase CAS 9001-92-7

383-02281 DISPASEⅡ 分散酶Ⅱ 1 g 9001-92-7
386-02271 DISPASE®Ⅰ 分散酶 10000 PU×6 9001-92-7

Roche公司的Dispase分散酶产品

货号 品名 规格
Roche-04942078001 Dispase® II (neutral protease, grade II) 1 g
Roche-04942078001 Dispase® II (neutral protease, grade II) 100mg

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Wako和光纯药抑制剂 Inhibitor

Wako和光纯药抑制剂 Inhibitor

上海金畔生物代理Wako和光纯药全线产品,欢迎新老客户访问Wako和光纯药官网或者咨询我们获取更多相关产品信息。

Wako和光纯药代理

货号 品名 规格
307-50771 α-Amylase Inhibitor, From Wheat  α-淀粉酶抑制剂,来源于小麦 1MG
335-40623 Antipain 25MG
333-40624 Antipain 100MG
339-40621 Antipain 0.5MG
253-00471 YC-1 5MG
244-00721 Xestospongin C 100UG
210-00921 U-73343 5MG
213-00911 U-73122 5MG
211-01051 U0126 5MG
208-09223 Trypsin Inhibitor, from Soybean
胰蛋白酶抑制剂,大豆来源
1G
204-09225 Trypsin Inhibitor, from Soybean
胰蛋白酶抑制剂,大豆来源
5G
202-09226 Trypsin Inhibitor, from Soybean
胰蛋白酶抑制剂,大豆来源
500MG
202-09221 Trypsin Inhibitor, from Soybean
胰蛋白酶抑制剂,大豆来源
100MG
204-11991 Trichostatin A
曲古柳菌素A
5MG
200-11993 Trichostatin A
曲古柳菌素A
1MG
206-15471 Trichodion 100UG
209-14481 N-Tosyl-L-phenylalanine Chloromethyl Ketone(TPCK) 250MG
205-14483 N-Tosyl-L-phenylalanine Chloromethyl Ketone(TPCK) 1G
208-09181 (+/-)-alpha-Tocopherol Nicotinate
alpha-维生素E烟酸酯
5G
206-09182 (+/-)-alpha-Tocopherol Nicotinate
alpha-维生素E烟酸酯
25G
207-15641 TMP-153 500MG
164-17321 DL-threo-PPMP Hydrochloride
D,L-苏式-PPMP盐酸盐
25MG
161-17331 DL-threo-PPMP Hydrochloride
D,L-苏式-PPMP盐酸盐
50MG
206-10351 1-(2-Tetrahydrofuryl)-5-fluorouracil
1-(2-四氢呋喃基)-5-氟尿嘧啶
1G
202-10353 1-(2-Tetrahydrofuryl)-5-fluorouracil
1-(2-四氢呋喃基)-5-氟尿嘧啶
5G
209-12041 Tautomycin
泰托霉素
100UG
198-10281 Swainsonine
八倾吲嗪三醇
1MG
196-12703 Sulindac
舒林酸
50G
190-12701 Sulindac
舒林酸
10G
195-12491 Sulfuretin
硫黄菊素
20MG
333-31091 SUC-GLY-PRO-MCA 5MG
549-00286 STREPTOZOTOCIN 5G
543-00284 STREPTOZOTOCIN 1G
545-00283 STREPTOZOTOCIN 500MG
549-00281 STREPTOZOTOCIN 100MG
198-08515 Streptomycin Sulfate
硫酸链霉素
500G
192-08513 Streptomycin Sulfate
硫酸链霉素
100G
194-08512 Streptomycin Sulfate
硫酸链霉素
25G
196-08511 Streptomycin Sulfate
硫酸链霉素
5G
197-10251 Staurosporine
星形孢菌素
100UG
193-10253 Staurosporine
星形孢菌素
500UG
191-11533 Spectinomycin Dihydrochloride Pentahydrate
盐酸壮观霉素
1G
195-11531 Spectinomycin Dihydrochloride Pentahydrate
盐酸壮观霉素
5G
193-12051 Simvastatin
新伐他丁
25MG
194-13561 γ-Secretase Inhibitor X 1MG
188-01593 Roscovitine, 98.0+ % (HPLC)
细胞周期蛋白B激酶抑制剂
10MG
182-01591 Roscovitine, 98.0+ % (HPLC)
细胞周期蛋白B激酶抑制剂
1MG
181-01723 Resveratrol
白藜芦醇
500MG
185-01721 Resveratrol
白藜芦醇
100MG
183-01901 Radicicol
根赤壳菌素
1MG
165-20281 Protease Inhibitor Mixture-DMSO Solution for Fungal and Yeast Extracts 1ML
161-11493 Prednisolone
强的松龙
5G
165-11491 Prednisolone
强的松龙
1G
162-19821 Pravastatin Sodium Salt
普伐他汀
25MG
168-19823 Pravastatin Sodium Salt
普伐他汀
100MG
163-20341 PP-3
PP3酪氨酸激酶抑制剂
1MG
166-20331 PP 2, 94.0+ % (HPLC) 1MG
166-20294 Piroxicam
炎痛喜康
10G
162-20291 Piroxicam
炎痛喜康
1G
168-20293 Piroxicam
炎痛喜康
5G
160-17781 Phloretin, 98.0+ % (HPLC)
根皮素
250MG
160-11181 Pepsinostreptin
抑胃酶素
10MG
163-20101 PACOCF3
1,1,1-三氟-2-十七烷酮
10MG
144-07331 NS-398
COX-2选择性抑制剂NS-398
5MG
140-07333 NS-398
COX-2选择性抑制剂NS-398
25MG
147-07343 Niflumic Acid
尼氟酸
250G
141-07341 Niflumic Acid
尼氟酸
50G
145-06381 Nicardipine Hydrochloride, 99.0+ % (HPLC)
盐酸尼卡地平
1G
141-06383 Nicardipine Hydrochloride, 99.0+ % (HPLC)
盐酸尼卡地平
5G
145-06761 NF-κB inhibitor SN50 500UG
147-07201 (S)-(+)-Naproxen
(S)-(+)-萘普生
5G

Wako 014-18331 Acibenzolar-S-methyl Standard 噻二唑素-S-甲基标准品

Wako 014-18331 Acibenzolar-S-methyl Standard 噻二唑素-S-甲基标准品

英文名称:Acibenzolar-S-methyl Standard
中文名称:噻二唑素-S-甲基标准品
品牌:Wako
CAS No.:135158-54-2
储存条件:2-10℃(RT)
纯度:99.0+% (qNMR)

其他农残兽残分析相关产品:

施用于作物上的农药,其中一部分附着于作物上,一部分散落在土壤、大气和水等环境中,环境残存的农药中的一部分又会被植物吸收。残留农药直接通过植物果实或水、大气到达人、畜体内,或通过环境、食物链最终传递给人、畜。

到目前为止,世界上化学农药年产 量近200万吨,约有1000多种人工合成化合物被用作杀虫剂、杀菌剂、杀藻剂、除虫剂、落叶剂等类农药。农药尤其是有机农药大量施用,造成严重的农药污染问题,成为对人体健康的严重威胁。

农药残留对健康的影响

食用含有大量高毒、剧毒农药残留引起的食 物会导致人、 畜急性中毒事故。长期食用农 药残留超标的农副产品,虽然不会导致急性中 毒,但可能引起人和动物的慢性中毒,导致疾病的发生,甚至影响到下一代。

影响农业生产

由于不合理使用农药,特别是除草剂, 导致药害事故频繁, 经常 引起大面积减产甚至绝产,严重影响了农业生产。土壤中残留的长残 效除草剂是其中的一个重要原因。

影响进出口贸易

世 界各国,特别是发达国家对农药残留问题高度重视, 对各种农副产品 中农药残留都规定了越来越严格的限量标准。许多国家以农药残留限量为技术壁垒,限制 农副产品进口,保护农业生产。2000年,欧共体将氰戊菊酯在茶叶中的残留限量从 10毫克/千克降低到0.1毫克/千克,使中国茶叶出口面临严峻的挑战。

农残限量

世界卫生组织和联合国粮 农组织(WHO/FAO)对农药残留限量的定义为 , 按照良好的农业生产(GAP)规范,直接或间接使用农药后,在食品和饲料中形成的农药残留物的最大浓度。目前,中国已制定了79种农药在32种(类 )农副产品中197项农药最高残留限量 (MRL)的国家标准。

农药标准品 (一)

农残专用级别或HPLC级别的农药标准品, 纯度均大于98%。

产品编号 产品名称 中文名称 CAS NO. 包装
205-16281 beta-Trenbolone Standard 三烯酮标准品 10161-33-8 200mg
020-15311 Butylhydroxyanisole Standard (mixture of isomers) 丁基羟基茴香醚标准品 25013-16-5 200mg
086-08241 Hydrocortisone Standard 氢化可的松标准品 50-23-7 200mg
158-02531 Oxacillin Sodium Monohydrate Standard 苯唑西林钠一水合物标准品 7240-38-2 200mg
159-2561 Oxydozanide Standard 五氯柳胺标准品 2277-92-1 200mg
208-16271 Trenbolone Acetate Standard 乙酸去甲雄三烯醇酮标准品 10161-34-9 200mg
159-02681 (5Z)-Orysastrobin Standard (5z)-肟醚菌胺标准品   50mg
011-20051 (Aminomethyl)phosphonic Acid Standard 氨甲基磷酸标准品 1066-51-9 200mg
044-26063 (E)-Dimethylvinphos Standard E-甲基毒虫畏标准品 71363-52-5 50mg
063-04131 (E)-Ferimzone Standard E-嘧菌腙标准品   200mg
139-15891 (E)-Metominostrobin Standard (E)-苯氧菌胺标准品 133408-50-1 100mg
132-15521 (E)-Mevinphos Standard (E)-速灭磷标准品 298-01-1 100mg
167-16691 (E)-Pyrifenox Standard E-啶斑肟标准品 83227-22-9 200mg
166-19841 (E)-Pyriminobac-methyl Standard 嘧草醚标准品 147411-696 200mg
215-01331 (R)-Uniconazole Standard 烯效唑标准品 83657-16-3 50mg
023-12741 (RS)-s-Butylamine Standard (+/-)-1-甲基丙胺标准品 13952-84-6 200mg
045-25231 (Z)-DimethyMnphos Standard (Z)二甲基亚硝胺标准品 67628-93-7 200mg
067-05011 (Z)-Fenpyroximate Standard (z)-唑螨酯标准品 149054-53-5 20mg
066-04121 (Z)-Ferimzone Standard (Z)-嘧菌腙标准品 89269-64-7 200mg
139-15911 (Z)-Metominostrobin Standard (z)-苯氧菌胺标准品 133408-51-2 20mg
139-15531 (Z)-Mevinphos Standard (Z)-速灭磷标准品 33845-4 100mg
160-16701 (Z)-Pyrifenox Standard (Z)-比芬诺标准品 83227-23-0 200mg
163-19851 (Z)-Pyriminobac-methyl Standard (z)-嘧草醚标准品 147411-709 50mg
044-29601 1.1-DiChloro-2.2-bis(4-ethylphenyl)Ethane Standard l,1-二氯-2,2-二(4-乙苯)乙烷标准品 72-56-0 200mg
054-04121 1 2-DIbromoEthane Standard Solution l,2-二溴甲烷标准溶液 106-93-4 1mLx5
133-14831 1-Methylpiperidine Standard 1-甲基哌啶标准品 626-67-5 200mg
141-06501 1-Naphthylacetamide Standard 萘乙酰胺标准品 86-86-2 200mg
148-06511 1-Naphthylacetic Acid Standard a-萘乙酸标准品 86-87-3 200mg
208-11911 2,4,5-T Standard 2,4,5-T标准品 93-76-5 200mg
204-13451 2,4,5-T-butyl Standard 2,4,5-特丁基标准品 93-79-8 200mg
203-15481 2,4,6-Tnchlorophenol Standard 三氧苯酚标准品 32296 200mg
048-29741 2,4-DB Standard 2,4-DB标准品 94-82-6 200mg
045-25591 2,4-DiChloroaniline Standard 2,4-二氯苯胺标准品 554-00-7 200mg
164-18161 2,4-PA-butyl Standard 2,4-聚酰胺-丁基标准品 94-80-4 200mg
045-29371 2,6-Difluorobenzoic Acid Standard 2,6-二氟苯甲酸标准品 385-00-2 200mg
043-29811 2,6-Diisopropylnaphthalene Standard 2,6-二异丙基标准品 24157-81-1 200mg
011-08711 2-Aminobenzimidazole Standard 2-氨基苯并咪唑标准品 93432-7 200mg
169-17871 2-Phenylphenol Standard 邻苯基苯酚标准品 90-43-7 200mg
206-16951 3-(2,4,6-TrimethylphenylsuIfonyl)-1,2,4-triazole Standard 3-(2,4,6三甲基苯基磺酰基)-1,2,4三唑标准品 149591-20-8 S0mg
134-11941 3-(Methylphosphinico)propionic Acid Standard 3-(甲基膦酸基)丙酸标准品 15090-23-0 200mg
085-08571 3-Hydroxycarbofuran Standard 3-羟基呋喃丹标准品 16655-82-6 50mg
046-28441 4,4-Dimethyl-2-oxazolidinone Standard 4,4-双甲基-2-噁唑烷酮标准品 26654-39-7 200mg
030-19511 4-CPA Standard 对氧苯氧乙酸标准品 122-88-3 200mg
086-08501 4-Hydroxybiphenyl Standard 对羟基苯酚标准品 92-69-3 200mg
131-15731 4-Methyl-1,2,3 thiadiazole-5-Carboxylic Acid standard 4-甲基-1,2,3-噻二唑-5-甲酸标准品 18212-21-0 100mg
080-08521 5-Hydroxythiabendazole Standard 5-羟基噻苯咪唑标准品 948-71-0 20mg
022-15251 6-Benzylaminopunne Standard 6-苄氨基嘌呤标准品 1214-39-7 200mg
032-20561 6-Chloropicolinic Acid Standard 2-氯吡啶-6-羧酸标准品 4684-94-0 100mg
016-20361 Abamectin Standard 阿维菌素标准品 71751-41-2 200mg
018-18591 Acequinocyl Standard 灭螨醌标准品 57960_19_7 200mg
011-18601 Acequinocyl-hydroxy Standard 羟基灭螨醌标准品 57960-31-3 200mg
014-16491 Acetamiprid Standard 啶虫脒标准品 160430-64-8 200mg
013-20511 Acetochlor Standard 乙草胺标准品 34256-82-1 100mg
018-19451 Acibenzolar Acid Standard 阿拉酸式苯标准品 35272-27-6 100mg
014-18331 Acibenzolar-S-methyl Standard 阿拉酸式苯-S-甲基标准品 135158-542 200mg
010-20521 Aafluorfen Standard 三氟羚草醚标准品 50594-66-6 200mg
013-15741 ACN Standard ACN标准品 2797-51-5 200mg
018-16651 Acrinathrin Standard 氟丙菊酯标准品 101007-06-1 200mg
017-15521 Alanycarb Standard 棉铃威标准品 83130-01-2 200mg
018-17011 Allethrin Standard 烯丙菊酯标准品 584-792 200mg
019-20611 Allidocfilor Standard 二丙烯草胺标准品 93-71-0 100mg
015-09733 Alloxydim Sodium Standard 禾草灭标准品 55635-13-7 200mg
019-13641 Ametryn Standard 莠灭净标准品 834-128 200mg
011-14941 Amitraz Metabolite HydroChlonde Standard 双甲脒盐酸盐代谢物标准品 51550-40-4 200mg
015-09593 Amitraz Standard 双甲眯标准品 33089-61-1 200mg

Wako Y-27632 331752-47-7

Wako Y-27632 331752-47-7
品牌:Wako
品牌中文简称:和光纯药
CAS No.:331752-47-7
储存条件:-20°C

CultureSure® Y-27632 for Cell Culture
CAS RN® : 331752-47-7

030-24021 1mg
036-24023 5mg
034-24024 25mg
030-24026 100mg

CultureSure® 10mmol/l Y-27632 Solution, Animal-derived-free
for Cell Culture
CAS RN® : 331752-47-7
Comparison

035-24593 1mL
039-24591 300uL

Y-27632, MF
for Cell Culture
CAS RN® : 331752-47-7

259-00613 5mg

257-00614 25mg

Y-27632

ES、人类iPS细胞冷冻保存产品

本产品是具有选择性的强力ROCK(Rho-associated coiled-coil forming kinase/Rho结合酶)抑制剂。通过收缩ROCK信号传导系统的血管平滑肌,抑制癌细胞的浸润和控制细胞分化。2007年笹井等人对人类ES细胞培养时,做了许多因分散而抑制细胞死亡的报告。

近年发现,本产品的人类ES细胞、人类iPS细胞冷冻保存液在冷冻保存后解冻,向培养基中添加10 μmol/L,会大幅改善克隆形成率。同时,冷冻保存的ES细胞解冻后,同样用含本产品的培养基进行培养,也可以得到不错的形成率。

◆产品概述

外观:白色-浅黄色、结晶性粉末-粉末

含量(HPLC):98.0%以上

溶解性:水(2.5 mg/mL)

保存:-20℃避光保存(惰性气体密封)

CAS No.:331752-47-7

C14H21N3O・2HCl・H2O=338.27

◆特点

  •   高品质:纯度98.0%以上(HPLC)
  •   价格低廉

◆实验步骤

①hESCs 维持培养

②分离的hESCs在10 μmol/L Y-27632溶液中分裂

③10 μmol/Y-27632中单个hESCs缓慢冷冻

④37℃水浴快速解冻

⑤接种到含有Y-27632的hESCs维持培养基

Y-27632, MF

预防人ES·iPS细胞死亡

Y-27632, MF已于2015年9月注册原药等登记原簿(Master File)的培养基添加物。

和光按照自主规格,严格管理原料,实行制造工程及分析实验验证,确保品质质量。

和光纯药工业是API Corporation唯一认可的Y-27632制造贩卖商。

◆产品概述

外观:白色-浅黄色、结晶性粉末-粉末

含量(HPLC):98.0%以上

溶解性:合格

比旋光度[α]D20(c=1.0,CH3OH):+2.0~+10.0゜

内毒素:低于0.25 EU/mg

活菌数检测:低于20 CFU/g

支原体检测:合格

CAS No.:331752-47-7

C14H21N3O・2HCl・H2O=338.27

◆相关产品

Y-27632 细胞生物学用

CultureSure 低分子化合物

ES·iPS细胞研究用低分子化合物

StemSure Series

液体培养基·细胞培养试剂

相关产品

产品编号 产品名称 规格 包装
030-24021
036-24023
034-24024
CultureSure® Y-27632
本产品已经过支原体检测、内毒素检测、细胞毒性检测。
细胞培养 1 mg
5 mg
25 mg
197-16275 StemSure® D-MEM(High Glucose) with Phenol Red and Sodium Pyruvate 细胞培养 500 mL
197-17571
193-17573
StemSure® hPSC Medium Δ

※不含bFGF

细胞培养 100 mL
100 mL×4
197-16775 StemSure® Serum Replacement (SSR) 细胞培养 500 mL
198-15781 StemSure® 10mmol/L 2-Mercaptoethanol Solution(x100) 细胞培养 100 mL
195-15791 StemSure® 50mmol/L Monothioglycerol Solution(x100)
能与2ME同等使用的2ME替代品。非毒物。
细胞培养 100 mL
190-15805 StemSure® 0.1w/v% Gelatin Solution 细胞培养 500 mL
195-16031 StemSure® Freezing Medium 细胞培养 100 mL
199-16051
195-16053
StemSure® LIF, Mouse, recombinant, Solution 细胞培养 106 units
106 units×10
180-02991
186-02993
rBC2LCN-FITC [AiLecS1-FITC] 细胞染色 100 μL

100 μL×5

参考文献

  1. Uehata, M., et al.: Nature, 389, 990 (1997).
  2. Sakamoto, K., et al.: J. Pharmacol. Sci., 92, 56 (2003).
  3. Nishimaru, K., et al.: J. Pharmacol. Sci., 92, 424 (2003).
  4. Watanabe, K., et al.: Nat. Biotechnol., 25, 681 (2007).
  5. Martin-Ibanez. R., et al.: Hum. Reprod, 23, 2744 (2008).
  6. Classen A. D., et al.: Mol. Reprod. Dev., 76, 722 (2009).
  7. Kawamata, M., et al.: Proc. Natl. Acad. Sci. USA., 107, 14223 (2010).
  8. Ito, H., et al.: Liver Int., 32, 592 (2012).
产品编号 产品名称 产品规格 产品等级 产品价格
257-00511 Y-27632 1 mg 细胞生物学  
253-00513 Y-27632 5 mg 细胞生物学  
251-00514 Y-27632 25 mg 细胞生物学  
253-00591 5mmol/L Y-27632 Solution 300 μL 细胞培养  
259-00613 Y-27632, MF 5 mg 细胞培养  
257-00614 Y-27632, MF 25 mg 细胞培养  

 

品牌 产品编号 产品名称 等级 规格 CAS No.
Wako 和光纯药 039-24591 CultureSure® 10mmol/l Y-27632 Solution, Animal-derived-free  CultureSure® 10mmol/l Y-27632 溶液,无动物来源 for Cell Culture 300 ul 331752-47-7
Wako 035-24593 CultureSure® 10mmol/l Y-27632 Solution, Animal-derived-free Cell Culture 1 ml 331752-47-7
Wako 257-00511 Y-27632 for Cellbiology 1 mg 331752-47-7
Wako 253-00591 5 mmol/L Y-27632 Solution for Cell Culture 300 ul 331752-47-7
Wako 030-24021 CultureSure® Y-27632  选择性ROCK抑制剂 for Cell Culture 1 mg 331752-47-7
Wako 036-24023 CultureSure® Y-27632  选择性ROCK抑制剂 for Cell Culture 5 mg 331752-47-7
Wako 034-24024 CultureSure® Y-27632  选择性ROCK抑制剂 for Cell Culture 25 mg 331752-47-7
Wako 259-00613 Y-27632, MF  选择性强效ROCK抑制剂 for Cell Culture 5 mg 331752-47-7
Wako 257-00614 Y-27632, MF  选择性强效ROCK抑制剂 for Cell Culture 25 mg 331752-47-7
Wako 253-00513 Y-27632 for Cellbiology 5 mg 331752-47-7
Wako 251-00514 Y-27632 for Cellbiology 25 mg 331752-47-7

Wako 9011-18-1 Sodium Dextran Sulfate 5000 葡聚糖硫酸酯钠5000

Wako 9011-18-1 Sodium Dextran Sulfate 5000 葡聚糖硫酸酯钠5000

Sodium Dextran Sulfate 5000
葡聚糖硫酸酯钠5000
品牌:Wako
品牌中文简称:和光纯药
CAS No.:9011-18-1
储存条件:室温

外观 White – slightly yellow, crystalline powder – powder

含量 Total sulfur : 15.0 – 20.0%

Sulfated product of dextran, which is a polymer of glucose, a polysaccharide. The average molecular weight is ca 5000 (range: 1000 to 9000). The product is used for enhancement of nucleic acid hybridization. As a reagent of the molecular biology grade, it has been confirmed for DNase and RNase activities.

葡聚糖的硫酸化产物,它是葡萄糖的一种聚合物,是一种多糖。 平均分子量约为5000(范围:1000至9000)。该产物用于增强核酸杂交。 作为分子生物学级的试剂,已证实其DNase和RNase活性。
别名:
Sodium dextran sulfate
Dextran sulfate sodium salt
Dextran sulfate, sodium salt
Dextran sulfuric acid ester sodium salt
Dextran Sulfate . Na
Dextran sodium sulfate
Dextran hydrogen sulfate sodium salt
Dextransulfuric acid ester sodium salt
Dextran Sulfate, NA
Dextran Sulphate Sodium Salt
其他相关产品:
品牌 产品编号 产品名称 等级 规格 CAS No.
Wako   和光纯药 197-09984 Sodium Dextran Sulfate 500,000  葡聚糖硫酸钠 500,000 for Biochemistry 100 g 9011-18-1
Wako   和光纯药 193-09981 Sodium Dextran Sulfate 500,000 葡聚糖硫酸酯钠盐 500,000 for Biochemistry 10 g 9011-18-1
Wako   和光纯药 191-08365 Sodium Dextran Sulfate 5,000  葡聚糖硫酸钠 5,000 for Biochemistry 500 g 9011-18-1
Wako   和光纯药 194-14921 Sodium Dextran Sulfate 36,000~50,000  葡聚糖硫酸钠 36,000~5,0000 No grade 10 g 9011-18-1
Wako   和光纯药 194-13402 Sodium Dextran Sulfate 5000  葡聚糖硫酸钠盐5,000 for Molecular Biology 25 g 9011-18-1
Wako   和光纯药 198-13405 Sodium Dextran Sulfate 5000  葡聚糖硫酸酯钠5000 for Molecular Biology 500 g 9011-18-1
Wako   和光纯药 199-09983 Sodium Dextran Sulfate 500,000  葡聚糖硫酸钠500,000 for Biochemistry 50 g 9011-18-1
Wako   和光纯药 196-13401 Sodium Dextran Sulfate 5000  葡聚糖硫酸钠盐5,000 for Molecular Biology 100 g 9011-18-1
Wako   和光纯药 197-08362 Sodium Dextran Sulfate 5,000  葡聚糖硫酸钠盐5,000 for Biochemistry 25 g 9011-18-1
Wako   和光纯药 199-08361 Sodium Dextran Sulfate 5,000  葡聚糖硫酸钠盐5,000 for Biochemistry 100 g 9011-18-1
Wako   和光纯药 196-14925 Sodium Dextran Sulfate 36,000~50,000  葡聚糖硫酸钠 36,000~5,0000 No grade 500 g 9011-18-1
Wako   和光纯药 190-14923 Sodium Dextran Sulfate 36,000~50,000 No grade 100 g 9011-18-1

Wako TRAP/ALP染色试剂盒

Wako TRAP/ALP染色试剂盒

英文名称:TRAP/ALP stain kit
TRAP/ALP双重染色试剂盒
品牌:Wako
品牌中文简称:和光纯药
储存条件:-20℃
等级:for Pathology Research

正常的骨代谢是通过成骨细胞生长与破骨细胞建立骨吸收而维持平衡。成骨细胞的标记酶是碱性磷酸酶(ALP)。破骨细胞的标记酶是抗酒石酸酸性磷酸酶(TRAP)。这两种标记酶在组织切片或者培养细胞过程中,被作为成骨细胞、破骨细胞存在的标记指标。

  本试剂盒可通过骨组织切片以及培养细胞中TRAP/ALP酶活性进行组织染色。通过观察成骨细胞和破骨细胞的染色成像,可检查细胞的分化状态和骨组织的分布情况。

◆特点

 ● 使用时混合3种溶液,可制备TRAP酶活性染色的显色底物溶液。

 ● ALP酶活性染色使用的是预混物溶液,操作简便。

 ● TRAP的活性部位呈红紫色,ALP的活性部位呈蓝色(茶褐色),可双重染色。

 ● 适用于骨组织切片(脱钙GMA树脂包埋切片)以及培养细胞。

◆试剂盒组成

1214815164846981.jpg

● 酒石酸溶液(×10)·······3mL
● 酸性磷酸酶底物液A······30 mL
●  酸性磷酸酶底物液B······3 mL
● 核染色试剂··············10 mL
● 碱性磷酸酶预混物溶液····30 mL

<备注>本产品可对应培养细胞包装24孔板-5次,96孔板-6次。

骨组织载玻片(一个载玻片约使用500μL)包装为60个。

培养细胞TRAP/ALP酶活性染色案例

● 用TRAP染色剂使破骨细胞呈红色
● 用ALP染色剂使成骨细胞的细胞膜、软骨细胞和细胞间膜呈茶褐色
● 用核染色试剂使各种细胞核呈蓝绿色。

图片3.jpg

RAW264细胞的TRAP活性染色

图片4.jpg

MC 3T3-E1细胞ALP活性染色

石蜡切片骨相关酶(TRAP,ALP)双重染色

东京大学医学部附属医院 整形外科脊椎外科 第2研究室 河源 元

1.前言

  检测相关细胞的生理活性是掌握生物体骨代谢状态的有效方法。骨组织使用碱性磷酸酶(ALP)进行染色可明确成骨细胞的成骨潜能,使用抗酒石酸酸性磷酸酶(TRAP)染色可明确破骨细胞的骨吸收能力。并且在同一切片上进行二重染色时,可同时识别二者。另外,本产品还具有使用脱钙标本进行二重染色实验,无需特殊机器,在一般的设施上即可观察骨代谢等特点。在这样的背景下进行二重染色的研究。

2.标准标本的制作

  用之前使用的树脂包埋法或冷冻切片法制成的标本作为标准标本,和脱钙石蜡切片相比。即酒精固定小鼠肘关节,用乙二醇甲基丙烯酸树脂包埋,硬组织切片机制作2μm切片,然后进行TRAP和ALP染色(图1)2)。再制作脱钙冷冻切片(图2)作为标准标本。然后,研究或改良固定、脱钙、染色等各阶段双重染色的可行性。

1.jpg

图3.本实验标准标本——小鼠肘关节染色标本。

左图为TRAP染色,右图为ALP染色。下图是二者的双重染色

染色效果均佳。以此作为阳性对照,与脱钙石蜡切片一起染色,进行染色性评估。

2.jpg

图2.脱钙冷冻切片酶染色—将柠檬酸脱钙的小鼠腰椎

和右上肢浸泡在30%蔗糖溶液,后经OCT复合包埋,

Tissue‐tekPINO冻结,Cryo3制作5μm冷冻切片。再对切片进行酶染色。

不仅可进行TRAP染色(左:红色)和ALP染色(中:褐色)的单染色,双重染色(右)也呈阳性。

3.固定

  ALP染色使用不能用福尔马林固定的新鲜材料,如果未在短时间内固定,会导致酶失活,因此推荐80%乙醇固定。①小鼠膝关节不进行一次固定,直接用乙醇直接浸泡进行二次固定组。②小鼠腰椎用福尔马林(4%多聚甲醛溶液)固定16小时后再用乙醇进行二次固定。③小鼠腰椎用福尔马林固定4天后进行二次固定。④临床案例也用福尔马林固定1个月后,再进行二次固定。对上述四种类型进行TRAP·ALP双重染色,研究染色的可行性。图3是各染色性的研究结果。本实验证明福尔马林固定从16小时到4天时间范围内均可进行TRAP·ALP双重染色。

3.jpg

图3.福尔马林的1次固定的固定时间和TRAP/ALP的染色性研究

①是未经福尔马林固定,仅用酒精2次固定的样品,ALP染色呈强阳性。TRAP染色呈阴性。

②是福尔马林固定16小时和③是固定4天,TRAP/ALP的染色效果均佳。

并且,福尔马林固定1个月的临床案例(骨软骨瘤)中TRAP染色呈阳性,但不能ALP染色。

4.脱钙

  ALP酶是含有金属Zn蛋白。通过脱钙处理,酶的组成成分Zn被去除,导致酶失活。为弥补此缺点,需要添加ZnSO4,作为ALP活化剂。换言之,100mL脱钙液需添加0.4mL  1%ZnSO4,来补充Zn离子。并且,作为脱钙剂,在柠檬酸盐酸缓冲液添加Zn的同时,也可尝试添加相同螯合剂EDTA溶液进行研究。另外,也可研究能否在酸性脱钙剂(甲酸福尔马林溶液)进行酶反应。结果显示,脱钙液添加EDTA溶液后染色良好。反之,甲酸福尔马林溶液不能进行酶染色(图4)。脱钙方法利用超声波脱钙装置(Histra-DC,正常光)在8~16℃的低温环境下,连续操作3-6天,对样品进行脱钙处理。脱钙后用添加甘氨酸的巴比妥缓冲液(pH7.4)清洗,磷酸钙附着在组织上,防止沉淀。

4.jpg

图4.脱钙液的种类不同可否双重染色

左图是选用酸性脱钙溶液中对组织伤害较少的甲酸福尔马林

脱钙溶液脱钙3天(Histra-DC,8~16℃)后的大鼠膝关节。

(Histra-DC,8-16℃)TRAP和ALP均不能染色。相对来说,中图、右图均可进行TRAP/ALP双重染色。

    染色法是Lorch的Gomori法。本次用的是偶氮染料法和耦合法结合的TRAP/ALP染色试剂盒(Wako,产品编号:294-67001)。Lorch推荐切片厚度为8μm,同时也研究了普通的4μm切片是否能染色、双重染色的顺序应该先染TRAP和ALP中的哪一个、封片剂是否必须选水溶性封片剂等问题。

5.染色

  染色法结果:偶氮染料法中切片过厚导致酶扩散图像。即用TRAP染色骨质有红染倾向,但即使是4μm厚度,只要增强反应条件(反应温度和反应时间),也能充分染色的。换言之,TRAP染色中反应温度从室温调至37℃,反应时间从30分钟调至45分钟或60分钟。ALP染色在37℃反应45分钟至3小时,或者室温(10-15℃)下反应时间增加至一晚。此结果显示,两者色调平衡,染色效果好(图5)。但是,反应条件的增强导致ALP染色切片产生了很多色素颗粒(图2)。另外,TRAP和ALP的染色顺序哪一个先染色都是可以的。如果先进行ALP染色时,在TRAP阳性部位呈明显的红色,鲜艳处为破骨细胞。获得比较好的标本。但是,ALP的反应产物经过TRAP溶液处理后,会产生白色混浊颗粒,沉淀在组织上。因此,先TRAP染色,接着ALP染色的方法是可行的。封装法是利用甲基绿数秒间核染色处理。水洗后,37℃下干燥,二甲苯浸透,用马里醇(Marinol)永久封存。有文章推荐在浸透前用推荐使用酒精脱水,但是会产生反应产物的溶解、扩散。

5.jpg

图5.脱钙石蜡包埋切片的TRAP/ALP双重染色

用1年龄小鼠腰椎的EDTA脱钙石蜡切片,进行TRAP/ALP双重染色。

TRAP阳性将破骨细胞染成红色,ALP阳性将细胞活性强的细胞(成骨细胞、软骨细胞)染成褐色。

(上:弱扩大,下:强扩大)


TRAP和ALP的双酶染色实验中通过对固定、脱钙、染色进行多处改良,实现了对脱钙石蜡标本的染色。但是酶扩散图像对TRAP染色的效果极佳。出于各种原因的考虑,关于固定步骤的影响,如果固定不充分的话,容易抑制脱钙水平。进而导致酶扩散的发生。另外,注意脱钙本身的影响也是有必要的。总而言之,和非脱钙标本相比较,脱钙标本的扩散图像更加明显。脱钙会是酶扩散变强。另外,我们将在今后对切片的厚度和二重染色顺序的影响进行着重研究。
6.

总结

7.产品列表

产品编号 产品名称 规格 包装
294-67001 TRAP/ALP双重染色试剂盒

TRAP/ALP Stain Kit

病理研究用 60次

 

【参考文献】

[1] 須田立雄、小澤英浩、高橋栄明著:「骨の科学」(医歯薬出版)(1985).

[2] 河原元:技術講座、続・病理組織標本作製完全マニュアルシリーズ第2回硬組織検査法,

   MedicalTechnology,24(12),カラーアトラス、1213-1222(1996).

[3] 「骨形態計測ハンドブック」第1版,p.67(1983)

[4] 末吉徳芳ら:技術講座、組織固定法—より良い固定を目指して,MedicalTechnology,

   37(8),857-864(2009).

[5] Localization of AlkalinePhosphatase in MammalianBones by I. JOANLORCH

[6] Conyers,R.A.J.:Biochem.Biophys.Acta(Amst),138,363-371(1976)

[7] Gomori,G.:Proc.Soc.Exptl.Biol.Med.,42,23(1939)

[8] Barka,T.and Anderson,P..:J.Histochem.Cytochem.10,741(1962)

[9] 影山圭三、渡辺陽之輔:「病理組織標本の作り方」,慶応義塾大学医学部病理学教室編,

   第5版(医学書院)(1986).