MS603 MitoProfile Blue Native OXPHOS
Complexes Detection Kit


300 g Kit: $575.00
600 g Kit: $945.00
Please contact us for volume discounts.


Brief Description & Uses

Blue Native polyacrylamide gel electrophoresis, BN-PAGE (1), is a simple and effective way to subfractionate mitochondrial proteins as intact complexes on a single gel (in one dimension). It can be used to detect altered assembly of these complexes arising from mutations in subunits, mutations in assembly factors, or mtDNA depletion. This type of analysis has been performed with biopsy samples, platelets and fibroblast cells from patients with suspected mitochondrial diseases. For a detailed protocol for BN-PAGE click here.

In this method multisubunit enzymes bind a charged dye Coomassie brilliant blue which allows their electrophoretic separation in the first dimension by the size of the complex. Complexes I-V with masses ranging from 950K to 200K are well resolved in the first dimension. The separated proteins can then be transferred to nitrocellulose membrane/PVDF by electrophoresis and Complexes I, II, III, IV and V can be detected by mAbs against CI-NDUFA9 (MS111), CII-70 kDa subunit (MS204), CIII-Core protein 2 (MS304), CIV-subunit IV (MS407) and CV-α subunit (MS507) respectively. Such one dimensional gels, are best analyzed by using single mAbs against each complex. Other Complex I mAbs are available for BNPAGE, specifically anti-GRIM-19 (MS103) and anti-20 kDa (MS105). A sample of purified bovine heart mitochondria can be supplied with these antibodies to act as a BNPAGE control sample upon request.

Sometimes a greater separation of enzymes is necessary - it is possible to separate the proteins within each individual complex. To do this, blotting is NOT performed after the first (NATIVE) dimension, instead gels are turned 90 degrees and run in a perpendicular second dimension which is denaturing (NON-NATIVE). In this way the protein subunits within each complex are separated. MitoSciences provides an optimized pre-mixed cocktail of the mAbs to SIMULTANEOUSLY detect Complexes I-V after 2nd dimension blotting (specifically the cocktail contains MS111, MS204, MS304, MS407 and MS507). An example of this kind of analysis in fibroblasts is shown in the figure below, also see examples of the use of BN-PAGE in patient case studies (link below). For a detailed description of 1st and 2nd dimension BN-PAGE click here. This cocktail is supplied with a sample of bovine heart mitochondria to act as a BNPAGE control sample.



Click here to review case studies for this product.


Specifications

Product: MitoProfile Blue Native OXPHOS Complexes
Detection Kit

Kit Components:

Premixed 306 g cocktail containing the mAbs MS111, MS204, MS304, MS407 and MS507 (see Table below). BHM Control.

Catalog #: MS603
Lot #:
Host Species: Mouse
Concentration: 1.5 mg/ml in HEPES-Buffered Saline (HBS) with 0.02% azide as a preservative.
Purity: Near homogeneity as judged by SDS-PAGE. The antibodies were produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation.
Suggested Working Concentration: The antibody cocktail (1.5 mg/ml) should be diluted 250x to a final working concentration of 6 g/ml for Western blotting
Applications: Western blotting detection OXPHOS complex assembly after BNPAGE.
Antigen Species Cross-Reactivity: human, bovine and mouse
Storage: 4C
Country of Origin: USA



Kit Components

ANTIGEN CAT # CLONE ID AMOUNT
(g)
FINAL CONC.
(g/ml)
IMMUNOGEN
C-I-39 (NDUFA9) MS111 20C11B11B11 120 2 Bovine Heart Complex I
C-II-70 (Fp) MS204 2E3GC12FB2AE2 6 0.1 Bovine Heart Complex II
C-III-Core 2 MS304 13G12AF12BB11 60 1 Bovine Heart Complex II, III
COXIV MS407 20E8C12 60 1 Bovine Complex IV, holoenzyme
C-V-α MS507 15H4C4 60 1 Bovine Complex V



Product References

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References for Oxidative Phosphorylation

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Images

MS603 Western blotting results


Downloadable Information
MS603 Specifications


Additional Information
Kit Components
Product Images
Product References
Protein References


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