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Analysis Note

ControlPOSITIVE CONTROL:

Vinculin is present in all cell types.

Application

Research CategorySignaling

Western blot. 1:1000; ~117kDa band. Suggested blocking buffer is TBS-Tween with 2% BSA. Suggested dilution buffer is TBS-Tween with 0.05% sodium azide. Preferred gel percentage is 10% and/or 4-20% (or similar) gradient gel.

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). If necessary 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 (see application notes below).

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. Note that the muscle-specific isoform, meta-vinculin (MW 150 kDa) will co-precipitate.

Optimal working dilutions must be determined by the end user.

APPLICATION NOTES FOR MAB3574

IMMUNOCYTOCHEMISTRY

For cultured cells, use the following method: 1) wash cells twice with buffer "A" (100 mM Pipes (to make it dissolve, titrate with KOH), 0.5mM MgCl2, 0.1 mM EDTA, 0.01M EGTA (to make it dissolve, titrate with KOH), pH 6.9); 2) fix 15 min @RT (3.7% formalin, 0.2% Triton X-100, 2M Glycerol, prepared on buffer A); 3) wash 3 times with buffer "A"; 4) proceed with incubation with primary Abs, etc.

Research Sub CategoryCytoskeletal Signaling

Anti-Vinculin Antibody, clone VIIF9 (7F9) is an antibody against Vinculin for use in IP, WB, IC, IH(P).

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Immunogen

Crude smooth muscle extract from normal human adult uterus.

Legal Information

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Other Notes

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Physical form

Liquid in 0.02M Phosphate buffer with 0.25M NaCl and 0.1% sodium azide.

Format: Purified

Quality

Tested

Specificity

Vinculin. Cellular localization is Intracellular, associated with cytoskeleton.

Storage and Stability

Maintain at 2-8°C in undiluted aliquots up to 6 months after date of receipt