The Msr family are well conserved proteins that function to reduce oxidized methionine to L-methionine. The SelR (MsrB) protein was identified as the enzyme responsible for reduction of the MICAL-oxidized Met 44 and Met47 amino acids of actin.. As part of the MOXtrue™ product line,human MSRB2 protein (MB201) [accession # Q9Y3D2 (MSRB2_HUMAN)] has been produced and purified from a bacterial expression system. The recombinant protein is N-terminally 6x Histidine tagged and is comprised of amino acids 42-182. Validation studies have shown that MB201 can reduce MICAL-oxidized actin which will enhance subtilisin A cleavage by >90% in limited proteolysis assays. MB201 has also been validated for the production of reduced actin which can be used in downstream applications such as sedimentation assays and actin polymerization assays.
To learn more about using MsrB2 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.
MsrB2 Protein Purity Determination
A 10 μg sample of MsrB2 protein 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 MsrB2 Treated Oxidized Actin MICAL-oxidized actin (MetO Actin) (Cat# MXA95) was diluted to 0.1 mg/ml (2.3 μM) and left untreated, or treated with DTT (10 mM), MB201 (0.3 μM) , or a combination of both (see method). 2 mg 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 |
Oxidized Actin Sedimentation +/- MsrB2 Treatment
MICAL-oxidized actin (MetO Actin) (Cat. # MXA95) 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 or 2x polymerization buffer supplemented with MB201 + DTT for 1 h 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
Oxidized Actin Polymerization +/- MsrB2 Treatment
Pyrene-labeled actin (Cat # AP05) and MICAL-oxidized pyrene labeled actin (Cat. # MPAX1) were diluted to 0.1 mg/ml (2.3 μM) (see method). Samples were then incubated with 2x polymerization buffer supplemented with nothing (labeled Control), DTT alone (labeled +DTT), MB201 0.3 µM + DTT (labeled +MB201 0.3), or MB201 1.2 µM + DTT (labeled +MB201 1.2),. Upon actin polymerization fluorescence was detected with a spectrophotometer (see method below). A.U. = arbitrary units
Click here for a detailed method
For more information contact: signalseeker@cytoskeleton.com
Associated Products:
MOXtrue™ 6xHis MICAL-1 Protein (Cat. # MIC01)
MOXtrue™ MICAL-oxidized Actin (Cat. # MXA95)
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: Lot 015
For the most recent publications citing this and other Signal-Seeker™ products, see our Signal-Seeker™ Validation Data Page click here
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If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
Cytoskeleton公司成立于1993年，专注于生物化学和细胞过程研究中的纯化蛋白和便捷试剂盒开发与生产。公司提供药物筛选、信号转导、细胞骨架研究相关的系列试剂盒和产品，尤其以细胞骨架相关研究见长，既能满足于样品较少的科学研究，也可以用于小规模筛选研究和高通量大规模筛选研究。此外，公司还提供微管蛋白，肌动蛋白，小G蛋白，GAPs，GEFs等现有产品的药物筛选服务。
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