Anti-Filamin A Antibody, clone PM6/317

Filamin is a structural protein that forms flexible cross-links between two actin filaments. Filamin is a homodimer of polypeptide chains each joined to the other at one end with an actin binding site ath the other. It is present in smooth muscle, fibroblasts, platelets and lymphocytes.

Expected to cross-react with chicken, guinea pig and rabbit.

Recognizes unprocessed, full length Human filamin (actin-binding protein; 270-280 kDa) as well as the 190 kDa N-terminal calpain cleavage fragment of filamin (Aakhus, 1992). Following induction of apoptosis in U937 cells, the antibody recognizes 170, 150, and 120 kDa N-terminal cleavage fragments of the full-length form presumably resulting from cleavage by activated caspase-3 (Umeda, 2001).

Human platelet protein.

Immunoblotting:
1:1000-1:4000. Because of the large size of the unprocessed forms of filamin, 4-7% PAGE gels and proteinase inhibitors are recommended.

Immunofluorescence:
1:50-1:200 dilution from a previous lot was used. Suitable for staining both frozen and paraffin embedded tissues (at lower dilutions). Microwave-citrate buffer antigen retrieval method recommended for paraffin sections.

Immunoprecipitation:
A previous lot was used on immunoprecipitation. Suggested lysis buffer is PBS with 0.5% triton X-100 with proteinase inhibitors (note for full length filamin include calpain inhibitors). 5 microliters of antibody for every 300-500 μL of cell lysate (200-500 μg/mL total protein is suggested. Incubation is 1 hour RT or overnight 4C; Protein A/G agarose beads or rabbit anti-mouse secondary capture antibody is recommended for best recovery. 4-8% acrylamide gels are recommended for full length filamin or the 190 kDa fragement visualization.

Optimal working dilutions must be determined by end user.

Research Category
Cell Structure

Research Sub Category
Cytoskeleton

This Anti-Filamin A Antibody, clone PM6/317 is validated for use in IF, IH, IH(P), IP, WB for the detection of Filamin A.

Routinely evaluated by Western Blot on Jurkat lysates.

Western Blot Analysis:
1:500-1:4000 dilution of this lot detected Filamin A on 10 μg of Jurkat lysates. Because of the large size of the unprocessed forms of filamin, 4-7% PAGE gels and proteinase inhibitors are recommended.

270-280 kDa & 190 kDa

Mouse monoclonal ascites IgG1 in buffer containing 0.1% sodium azide.

Unpurified

Stable for 1 year at -20°C in undiluted aliquots from date of receipt.
Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Control
Positive control tisse: skin, jurkat cell lysate.

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

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