- Overview
- Data/Specifications
- Literature/Support
- How It Works
- Related Products
Overview
Aggrecanases ADAMTS1, ADAMTS4 and ADAMTS5 are members of the ADAMTS-family of proteases (ADAMTS = A disintegrin and metalloprotease with thrombospondin motif). They aresynthesized as multidomain proteins consisting of prodomains, protease, disintegrin, thrombospondin and cys-rich domains followed by spacer regions and additional thrombospondin motifs. The prodomains are cleaved-off intracellularly and the enzymes are processed further from their C-terminal ends after secretion.
Aggrecanases cleave specific peptide bonds in aggrecan, the main proteoglycan of articular cartilage. They hydrolyze also other lecticanes as versican and brevican. Convincing evidence indicates that aggrecanases are mainly responsible for excessive aggrecan degradation in osteoarthritic joints.
Data/Specifications
Species: human
Sample Type: cell culture supernates
Sample Size: 100 uL
Standard Curve Range: 0.062 - 4 nM
Sensitivity: 0.025 nM
Assay Length:3.75 hours
Literature/Support
Product Insert:
Aggrecanase Activity Assay Insert (PDF)
Articles/FAQ:
ELISA Troubleshooting Guide
ELISA Data Reduction Guide
Measuring aggrecanase activity to identify potential therapeutics for osteoarthritis. (Blog Post)
How It Works
The Aggrecanase Activity Assay measures activity of aggrecanases. It is used to screen and characterize aggrecanase inhibitors. This assay consists of two modules, the Aggrecanase Module and the ELISA Module. A recombinant fragment of human aggrecan interglobular domain (aggrecan-IGD) is first digested with ag- grecanase. Proteolytic cleavage of the substrate releases an aggrecan peptide with the N-terminal sequence ARGSVIL (ARGSVIL-peptide). The ARGSVIL-peptide is then quantified with two monoclonal anti-peptide antibodies.
Aggrecanase module:Proteolysis of aggrecan-IGD by aggrecanase
Aggrecan-IGD is incubated with standard aggrecanase and samples of unknown aggrecanase activity. In addition to aggrecanase, different concentrations of aggrecanase inhibitors can be added. The reaction is then stopped by dilution with EDTA-containing buffer.
ELISA module: Aggrecan peptide ELISA
ARGSVIL-peptide standard, proteolytic digestions of aggrecan-IGD with standard aggrecanase and samples are incubated in microtiter wells precoated with anti-ARGSVIL-neoepitope antibody. ARGSVIL-peptide is bound to the coated antibody, while other components are removed by washing and aspiration. The bound ARGSVIL-peptide is detected with a second peroxidase-labeled antibody. Any excess of the conjugate is removed by washing and aspiration. The amounts of peroxidase bound to different wells are determined in reactions with peroxidase substrate TMB. The reactions are stopped by addition of sulfuric acid solution and absorbance is read at 450 nm in a microtiter plate spectrophotometer.
Related Products
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请大神前辈指点指点,已经研三了,实验很不顺利,再这样都要延期的节奏了。请问最近急性分离出来的大鼠心肌细胞很不耐钙,做膜片钳封接不上可能是什么原因造成的?谢谢了!
想用二步酶消法来分离毛乳头细胞,一个毛囊大概有多少个毛乳头细胞?处于生长期的毛囊在显微镜下有什么特点?先谢谢大神们指点
我做的临床试验,就想简单检测PBMC细胞的凋亡,上流式检测,有啥检测效果好的步骤推荐一下,谢谢
最近在做黄体化颗粒细胞分离培养,但是注射器吸取大卵泡液或者从下颗粒细胞离心时就会凝固成果冻状,大家有碰到过这样的事情么?已经好几次了,一直是这样,大家给支个招吧
经过无数次的大鼠肝脏插针灌注,忍受过实验结果不理想的打击和折磨,今天终于成功了,特分享些相关图片以鼓励还在奋战中的网友们。
我的步骤:
1、16-22天小鼠颈椎脱臼法处死后,75%酒精浸泡30秒消毒,睾丸取出后置预冷PBS,40分钟(路上所需时间)后开始处理。
2、剔除白膜,剪碎睾丸。0.25%胰酶(含edta)1.5ml/个睾丸37°C消化30-50分钟,等体积DMEM/F12含10%FBS终止消化,1000r/MIN离心5分钟,弃上液。
3、加0.1%胶原酶消化30分钟,过200目网筛,1000r/MIN离心5分钟,弃上液。
4、DMEM/F12含10%FBS5ml重悬后转如培养瓶,入孵育箱37°C5%CO2培养。
请教各位,我到底是哪里有问题,是胰酶消化的时间长了吗?
近期根据经验总结了原代肿瘤细胞分离培养的2个关键因素。
1、组织的活性:如果我们取的都是坏死的组织或者结缔组织,我相信任何方法都不可能分离出来活的细胞的。另外,取完的组织要冷藏存放,并且越早分离越好,最好在24h内,最久不要超过48h。
2、组织的消化:在组织消化前,组织剪的越碎越好(1mm3左右为宜),组织的大小决定了组织的消化时间,不同组织消化时间差别较大:例如肺癌在2h-2.5h;食管癌在1个多小时。
希望可以帮到各位研友,另外如果大家有更好的经验可以一起来讨论!
前辈们好,近期老板布置任务需要分离小鼠肝脏细胞,查了好多文献,都没有写胶原酶的cat#,只写sigma订购,想请教下,是否有人做过,可以提供下货号和使用方法吗?十分感谢。另外,文中还提到digitonin,如果有一起用到的话麻烦帮我查看下,谢谢。
大概步骤如下:c57小鼠大概六七只,酒精浸泡后不处死取出,迅速打开胸腔取出心脏放入冰PBS冲洗。待心脏取出完毕,冲洗移入有PBS青霉素小瓶中用眼科剪剪成较小组织块。
之后加入0.1%胰酶消化(之前浓度为0.25%,活细胞数量更少。)吹打1min.放入孵箱中3min.弃去上清,加入∥型胶原酶(0.1%)吹打10Min,放入孵箱15Min。取出后加入1ml胰酶。吹打1Min后加入含血清F12培养液终止消化,800转离心5Min。之后取沉淀重悬种板。90Min差速贴壁法纯化心肌细胞。
48h后只有少量(个位数)细胞贴壁,呈三角形。
第一次用0.25%胰酶时离心后有大量鼻涕样物质。
第二次胰酶浓度减小后没有,但细胞沉淀少,且为半透明微白。是不是离心时间不够。
感觉细胞整体状态都不好,几次都没有大量心肌细胞,更不用说观察到心肌细胞搏动。
老师们能不能帮忙分析一下是哪里的原因呢。
有没有比较稳定的小鼠乳鼠心肌细胞分离步骤呢。
希望大神快点出现。
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