Anti-FAP-alpha Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for FAP-alpha on Western blot?

FAP-alpha migrates at approximately 95-97 kDa on reducing SDS-PAGE, slightly above its predicted mass of 88 kDa due to N-glycosylation at multiple sites. The 760-amino acid polypeptide contains signal peptide, catalytic domain, and transmembrane anchor. You may observe additional higher-molecular-weight bands representing incompletely deglycosylated forms or dimers, as FAP-alpha can form homodimers in vivo. For sharpest bands, use freshly prepared lysates and avoid repeated freeze-thaw cycles. If working with secreted or shed FAP forms, expect slight mobility shifts depending on proteolytic cleavage at the membrane-proximal region.

Does this FAP-alpha antibody detect both endopeptidase and dipeptidase active forms?

This polyclonal antibody targets a broad epitope range and should recognize FAP-alpha regardless of enzymatic conformation, since the antigen spans residues outside the immediate active-site pocket. Both the post-proline endopeptidase and dipeptidyl peptidase activities reside in the same 760-amino acid polypeptide and depend on Ser624 catalytic triad residues. The antibody detects native, full-length FAP under denaturing conditions on Western blot. For functional assays distinguishing enzymatic states, pair immunoprecipitation with this antibody with substrate-cleavage activity assays using gelatin or synthetic fluorogenic dipeptides to confirm catalytic competence in your sample.

What starting dilution should I use for Western blot with this FAP-alpha antibody?

Begin at 1:1000 dilution in 5 percent non-fat milk or BSA in TBST for overnight incubation at 4°C. This dilution is optimized for standard mammalian cell lysates expressing physiological FAP-alpha levels. If signal is weak in low-expressing samples, titrate upward to 1:500. Conversely, in activated fibroblast or tumor stromal lysates where FAP expression is elevated, you may achieve clean blots at 1:2000. Include a positive control such as lysate from MRC-5 fibroblasts stimulated with TGF-beta or human pancreatic cancer-associated fibroblasts, which express robust FAP-alpha. Always run parallel no-primary controls to confirm specificity.

Is this antibody validated for mouse and rat FAP-alpha or only predicted cross-reactivity?

Human FAP-alpha is validated; mouse and rat are predicted cross-reactive based on sequence homology. Human and mouse FAP share approximately 88 percent amino acid identity, with conserved epitope regions in the extracellular catalytic domain. Rat homology is similarly high. However, we have not formally validated performance in mouse or rat lysates with side-by-side controls. If working with rodent samples, plan a pilot titration experiment and include a knockout or FAP-negative cell line as negative control. Some users report successful detection in mouse tumor stroma by IHC, but expect potential differences in affinity and optimal working dilution compared to human tissues.

What positive control tissue or cell line should I use for FAP-alpha detection?

Cancer-associated fibroblasts and activated stromal fibroblasts are the most reliable positive controls, as FAP-alpha expression is minimal in quiescent fibroblasts and most epithelial cells. Human pancreatic, breast, or colorectal tumor stroma typically show strong FAP immunoreactivity. For cell lines, consider MRC-5 or WI-38 lung fibroblasts treated with TGF-beta1 for 48-72 hours to upregulate FAP expression, or immortalized cancer-associated fibroblast lines if available. Avoid using whole-tumor homogenates without microdissection, as stromal FAP can be diluted by FAP-negative tumor parenchyma. Recombinant FAP-alpha protein serves as a sizing control but may not reflect glycosylation of the native antigen.

Can I use this FAP-alpha antibody for immunohistochemistry on formalin-fixed paraffin-embedded tissue?

Triple Point Biologics antibodies are validated for Western blot applications. For FFPE tissue, antigen retrieval is typically required because formalin crosslinks mask epitopes. Start with citrate buffer pH 6.0 heat-induced epitope retrieval, heating slides in a pressure cooker or steamer for 20 minutes. Test antibody dilutions between 1:100 and 1:500. FAP-alpha localizes to the cell membrane and cytoplasm of stromal fibroblasts, particularly prominent in desmoplastic tumor stroma and healing wounds. Include adjacent normal tissue as an internal negative control, and verify specificity with a blocking peptide or isotype control. Frozen sections generally yield stronger signal without retrieval.

How should I store this FAP-alpha antibody and what is the shelf life?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which denature immunoglobulins and reduce titer. The 100-microgram pack size is suitable for dividing into 10-20 microliter aliquots depending on your typical experiment scale. Rabbit polyclonal antibodies in glycerol-containing storage buffer remain stable for at least 12 months at -20°C. For short-term use within two weeks, you may hold working aliquots at 4°C with 0.02-0.05 percent sodium azide as preservative. Avoid storing in frost-free freezers where temperature cycling accelerates degradation. If precipitate forms after thawing, centrifuge briefly before use rather than vortexing vigorously.

Why do I see multiple bands on my FAP-alpha Western blot?

Multiple bands likely reflect glycosylation heterogeneity, proteolytic processing, or dimer formation. FAP-alpha is extensively N-glycosylated, producing a smear or ladder between 95-110 kDa. Treatment with PNGase F or Endo H will collapse bands to the core 88-kDa polypeptide. You may also detect a soluble 85-kDa form resulting from ectodomain shedding by membrane metalloproteases, particularly in conditioned medium samples. Homodimers around 190 kDa appear under non-reducing conditions. Lower-molecular-weight bands below 80 kDa usually indicate degradation; add protease inhibitor cocktail to lysis buffer and keep samples cold. If bands persist across multiple preps, confirm identity by peptide mass fingerprinting or siRNA knockdown.