Myosin Antibody (V/10 SM-M10) Summary
| Immunogen |
Crude smooth muscle extract from normal human adult uterus.
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| Specificity |
Smooth muscle myosin, heavy chain. By Western Blot the antibody recognizes a protein of 200-204 kDa.
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| Isotype |
IgG1
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| Clonality |
Monoclonal
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| Host |
Mouse
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| Gene |
MYH14
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| Purity |
Protein A or G purified
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Applications/Dilutions
| Dilutions |
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| Application Notes |
Western Blot (see application notes below). Suggested blocking buffer is TBS-Tween with 2% BSA. Suggested dilution buffer is TBS-Tween with 0.05% sodium azide. Preferred gel percentage is 5% (see application notes). Immunohistochemistry on frozen and paraffin embedded tissue sections. Suggested fixation for frozen tissue sections is acetone fix for 6 minutes at room temperature. For formalin fixed paraffin embedded tissue sections: microwave in 0.01M citrate buffer (pH 6.0) for 8-10 minutes (note that all microwaves differ and adjustments may need to be made) follow with enzyme digestion (0.01% pronase for 10 mintues). Suggested blocking agent is fetal bovine serum. The antibody has also been used successfully on methyl-Carnoy fixed tissue. Immunocytochemistry/Immunofluorescence Immunoprecipitation. Suggested extraction buffer is 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid-NaCl and 0.5 mM PMSF. Final reaction volume is 1 mL and suggested capture agent is agarose conjugated anti-mouse IgG. APPLICATION NOTES FOR MAB3572 WESTERN Blot To achieve good resolution of myosin heavy chain isoforms with distinct molecular weight (200 – 2004 kDa), the following procedure should be followed: 1). Pyrophoshate extraction buffer for sample preparation (see below); run SDS-PAGE in 5% gel. Important: for better resolution of the MHC bands, use electrophoretic buffer with pH 8.2 (i.e. 0.1 less than standard), and prepare resolving gel (5%) with pH 9.0 (not 8.8 as usual). Also help thorough degasing of the resolving gel mixture (H2O, acrylamide, EDTA, pH 9.0, before (!) adding SDS, TEMED and APS). Run SDS-PAGE longer than after the dye front runs off (use 200 kDa MW markers and let its 200 kDa band run at least to the middle of 8X8 gel (using a big size gel (not the mini-gel!) will enhance the quality of MHC band resolution). Pyrophosphate extraction buffer: (40mM Na4P2O7x10H2O, 1mM MgCl2, 1mM EGTA (add KOH to dissolve EGTA), PMSF, pH 9.5). To extract acto-myosin from tissues/cells, shake minced tissue or cells in cold extraction buffer 1 hr on ice bath (0oC), centrifuge @10,000g for 10 min at +2-8 oC, take supernatant and mix it 1:1 with standard Laemmli sample buffer, boil, run SDS-PAGE in 5% gel (see above).
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Packaging, Storage & Formulations
| Storage |
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
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| Buffer |
20mM Phosphate and 0.25M NaCl
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| Preservative |
0.1% Sodium Azide
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| Concentration |
1 mg/ml
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| Purity |
Protein A or G purified
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Notes
Relevant Entrez Gene Numbers: NM_152994.2
Alternate Names for Myosin Antibody (V/10 SM-M10)
- DFNA4
- FLJ13881
- FLJ43092
- KIAA2034DKFZp667A1311
- MHC16
- MYH14 variant protein
- MYH17
- Myosin heavy chain 14
- Myosin heavy chain, non-muscle IIc
- myosin
- myosin, heavy chain 14
- myosin, heavy chain 14, non-muscle
- myosin, heavy polypeptide 14
- myosin-14
- NMHC II-C
- NMHC-II-C
- Non-muscle myosin heavy chain IIc
- nonmuscle myosin heavy chain II-C