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
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