MsrB2 Protein aa: 42-182 (6xHis-tag)

On Arrival: 4°C

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.
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Each lot of purified protein is quality controlled to provide high batch to batch consistency, see COA documents.
MsrB2 Protein Purity Determination
A 10 ?g 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 Red^TMProtein 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 ?M) and left untreated, or treated with DTT (10 mM), MB201 (0.3 ?M) , or a combination of both (see method). 2mg 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.
Oxidized Actin Sedimentation +/- MsrB2 Treatment
MICAL-oxidized actin (MetO Actin) (Cat. # MXA95) was diluted to 0.2 mg/ml (4.6 ?M) or 0.8 mg/ml (18.4 ?M) (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.
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 ?M) (see method). Samples were then incubated with 2x polymerization buffer supplemented with nothing (labeled Control), DTT alone (labeled +DTT), MB201 0.3 uM + DTT (labeled +MB201 0.3), or MB201 1.2 uM + DTT (labeled +MB201 1.2), . Upon actin polymerization fluorescence was detected with a spectrophotometer (see method below). A.U. = arbitrary units
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