MICAL interacts with F-actin and uses NADPH as a cofactor to oxidize actin at Met44 and Met47 (b-actin nomenclature). Functionally, oxidation of Met44 has a profound effect on actin polymerization because the residue resides in the D-loop of subdomain 2 of the protein, which is critical for actin subunit contacts; thus, upon oxidation, Met44 becomes negatively charged and interferes with actin monomer-monomer interaction and promotes F-actin severing and depolymerization. Regulation of actin oxidation at Met44/Met47 has been shown to destabilize F-actin in vivo and to play a key role in a growing number of cellular processes. As part of the MOXtrue™ product line, rabbit skeletal muscle actin protein (MICAL-oxidized) (MXA95) has been enzymatically oxidized at methionines 44 and 47 with the MICAL flavoprotein monoxygenase protein. Purified MICAL-oxidized actin has reduced susceptibility to subtilisin A cleavage at M47/G48 by > 90%, and has also been validated for downstream applications such as sedimentation assays.
To learn more about using MICAL-oxidized Actin as a research tool see our datasheet
To learn more about the MICAL/MsrB/Actin physiological redox system see our Newsletter
Each lot of purified protein is quality controlled to provide high batch to batch consistency, see COA documents.
Actin Protein (MICAL-Oxidized) Purity Determination
A 50 μg sample of actin protein (MICAL-oxidized) was separated by electrophoresis in a 4- 20% tris-glycine gel and stained with Coomassie Blue. Protein quantitation was performed using the Precision RedTM Protein Assay Reagent (Cat. # ADV02). Mark12 standard molecular weight markers are from Invitrogen.
Subtilisin Assay on MICAL-Oxidized Actin vs Native Actin
Actin (Cat. # AKL99) and MICAL-oxidized actin (Cat. # MXA95) was diluted to 0.1 mg/ml (2.3 μM). 2 μg of each sample was then left untreated, or treated with subtilisin (1:200 w/w) for 15 min. Samples were then separated by SDS-PAGE and visualized with Coomassie staining.
Click here for a detailed method
Actin Sedimentation Oxidized Versus Native Actin
Actin (Cat# AKL99) and MIcal-oxidized actin was diluted to 0.2 mg/ml (4.6 μM) or 0.8 mg/ml (18.4 μM) (see method). Samples were then incubated with 2x polymerization buffer at room temperature. Samples were spun in an ultracentrifuge at 100,000 g for 1.5 h. Samples were then separated by SDS-PAGE and visualized with Coomassie staining.
Click here for a detailed method
For more information contact: signalseeker@cytoskeleton.com
Associated Products:
MOXtrue™ 6xHis MICAL-1 Protein (Cat. # MIC01)
MOXtrue™ 6xHis MsrB2 Protein (Cat. # MB201)
MOXtrue™ MICAL-oxidized Pyrene Labeled Actin (Cat. # MPAX1)
Rabbit Skeletal Muscle Actin (Cat. # AKL95)
Pyrene Labeled Rabbit Skeletal Muscle Actin (Cat.# AP05)
For product Datasheets and MSDSs please click on the PDF links below.
Certificate of Analysis: available upon request
For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here
Visit our Signal-Seeker™ Tech Tips and FAQs page for technical tips and frequently asked questions regarding this and other Signal-Seeker™ products click here
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
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【过敏性咳嗽血清学IGE检查】
以过敏患者血清作为实验材料的本外试验方法称为血清学试验。其它体液如炎症部位分泌物、渗出物、灌洗液也可采用相同的实验方法进行检测。主要检测项目有总IgE和特异性IgE，即过敏原特异性IgE。
【什么是总IgE？】
IgE即免疫球蛋白E，是I型变态反应病如过敏性鼻炎、过敏性哮喘、异位性皮炎、湿疹、急慢性荨麻疹发病机制中起主要作用的免疫分子，因而在过敏反应的免疫学实验诊断中是首选的检测项目。总IgE是过敏性疾病的特异性检查项目，IgE水平增高提示I型变态反应病的可能性大，但不能用于判断过敏原。
【IgE的特点】
IgE是血清浓度最低的免疫球蛋白 ，只有血清中IgG浓度的万分之一。IgE对热不稳定，是半衰期最短一的免疫球蛋白 ，只有2.8天，与细胞表面结合的IgE半衰期稍长，8~14天，IgE由变应原入侵部位（鼻咽、支气管、胃肠道）的黏膜固有层中的浆细胞合成。在各类免疫球蛋白中，IgE是合成率最低、分解率最高的。属于亲细胞抗体，过敏体质者的胎儿脐带血中IgE浓度可能升高，检测脐血中IgE浓度可用于评估胎儿过敏体质的可能性。
【IgE检测方法】
通常用ELISA方法检测总IgE。由于血清IgE浓度很低，一般酶免疫试验方法的敏感性不足以检出血清IgE，现在常规实验室检测血清IgE的试剂盒采用生物素——抗生物素蛋白 放大的ELISA。试剂盒中所含用于制定标准曲线的IgE标准品和检测结果的IgE浓度单位与其它免疫球蛋白 不同，不是用mg/L表示，而是用u/ml或ku/l表示。
【IgE的正常值（参考范围）】：
血清IgE水平在正常人群中呈偏态分布，即多数人为0或接近于0，IgE水平越高的人数越少。因此计算平均值时应计算几何平均值才能反映其真实情况，即用对数转换后其分布才能近似正态分布。
健康人群血清IgE水平与年龄关系较大，小儿和老年人的IgE水平低于成年人。新生儿血清中IgE水平很低，接近于零。随年龄增长，IgE水平也不断升高，5~7岁后接近正常人水平。按Pharmacia公司提供的参考范围，1个月以内<12KU/L,1岁<11KU/L,2~4岁<33KU/L,5岁以上至成人<85KU/L.
过敏性疾病患者的血清IgE水平可达2000~8000KU/L,当IgE水平高于2000KU/L时应考虑寄生虫感染.
有时血清总IgE水平检测结果为0或参考范围内低值,并不能排除过敏性疾病的可能,须结合临床表现和血清特异性IgE检测结果进行判断.
【什么是特异性IgE检测(sIgE）？】
通常所称的过敏原检测，并非真正检测血液样本中的过敏原分子，而是间接地检测其中针对某种过敏原的特异性IgE分子，特异性IgE检测实际上是检测过敏原特异性IgE，即检测样本中针对某种变应原的特异性IgE，从而间接地判断患者是否对某种过敏原过敏。
环境中常见的过敏原包括以下类别：
寄生虫和微生物：各种螨类（屋尘螨和粉尘螨等）、各种真菌（点青霉、烟曲霉、分枝孢霉、交连孢霉等）、蟑螂。
植物花粉：各种草花粉（豚草、葎草、蒿草）、各种树花粉（桑树、柏树、悬铃木、桦树、榆树、柳树、杨树等）。
动物皮毛：猫、狗、马、鸽子等动物的毛和皮屑。
