Anti-DPP-9 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for DPP-9 on Western blot?

Full-length human DPP-9 runs at approximately 98-100 kDa on Western blot, corresponding to the 863-amino acid protein (UniProt Q86TI2). You may observe minor gel-shift variability depending on buffer composition and acrylamide percentage. Post-translational modifications have not been extensively characterized for DPP-9, so additional bands are uncommon but possible in certain cell types. If you detect lower molecular weight species, consider proteolytic processing or degradation during lysis. Use fresh protease inhibitors and keep samples cold to preserve the full-length protein.

What starting dilution should I use for DPP-9 Western blot?

Start at 1:1000 dilution in blocking buffer for overnight incubation at 4°C, which is the validated dilution for this rabbit polyclonal. DPP-9 is a moderately abundant cytosolic protein in most hematopoietic cells, so signal should be detectable without concentration steps. If background is high, you can dilute to 1:2000 or reduce incubation time to 2 hours at room temperature. For low-expressing cell lines, try 1:500 first. Always include a lysate titration alongside antibody optimization to distinguish weak expression from suboptimal conditions.

Does this antibody cross-react with DPP4 or DPP8?

DPP-9 shares significant sequence homology with DPP4 and DPP8, so cross-reactivity is a legitimate concern. This polyclonal was raised against a region spanning residues within the DPP-9 sequence; however, specificity against closely related family members should be confirmed in your system. Run lysates from DPP4-high tissues like kidney or DPP8-expressing controls alongside DPP9-knockout or knockdown samples if available. Single bands at the correct molecular weight in appropriate cell types suggest specificity, but definitive validation requires genetic controls or blocking peptides when paralogs are co-expressed.

Which cell lines or tissues express high levels of DPP-9 for positive controls?

DPP-9 is broadly expressed but particularly abundant in immune cells. Human monocytes, macrophages, and THP-1 cells are reliable positive controls due to DPP-9's role in inflammasome suppression. Peripheral blood mononuclear cells freshly isolated or activated also show robust expression. For tissue lysates, spleen and lymph node typically yield strong signal. Avoid using DPP4-high tissues like kidney or intestine as your only control, since family cross-reactivity could confound interpretation. HEK293T cells express moderate DPP-9 and serve as a transfectable background for overexpression or knockdown experiments.

Can I use this DPP-9 antibody on non-human primate samples?

This antibody shows potential cross-reactivity with monkey DPP-9 based on sequence conservation, but it has not been formally validated in non-human primate lysates. Human and macaque DPP-9 share greater than 95% identity across most epitope regions, so cross-reactivity is probable. If you plan to work with cynomolgus or rhesus samples, run a pilot Western blot at 1:1000 dilution alongside human control lysate. Confirm that the band migrates at the expected molecular weight and that signal is absent in a knockout or pre-immune serum control before committing to full experiments.

What lysis buffer should I use to preserve DPP-9 protein integrity?

DPP-9 is a soluble cytosolic protein, so standard RIPA buffer with protease inhibitors works well. Use freshly added protease inhibitor cocktail and keep samples on ice throughout lysis and clarification. Because DPP-9 contains an active serine protease domain, include serine protease inhibitors like PMSF or AEBSF to prevent autoproteolysis or cleavage of binding partners. Avoid freeze-thaw cycles; aliquot lysates after quantification and store at -80°C. If you plan to study DPP-9 in complex with NLRP1 or CARD8, consider gentler non-denaturing buffers and co-immunoprecipitation-compatible detergents.

How should I store this DPP-9 antibody to maintain activity?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. After the first thaw, the working aliquot can be kept at 4°C with 0.02-0.05% sodium azide for up to one month if you use it frequently. Do not store diluted antibody in blocking buffer for more than a week; prepare fresh dilutions for critical experiments. If you notice increased background or weak signal over time, thaw a fresh aliquot and compare side-by-side. Rabbit polyclonal antibodies are generally stable for 12-24 months under proper storage conditions.

What is the functional difference between DPP-9 peptidase activity and inflammasome suppression?

DPP-9's dipeptidyl peptidase activity cleaves N-terminal dipeptides from proline- or alanine-containing substrates, but its most studied function is non-enzymatic: sequestering cleaved NLRP1 and CARD8 fragments to prevent inflammasome activation. Importantly, catalytic activity is required for the suppressive function, likely through proper folding or substrate engagement that stabilizes ternary complexes. Neither NLRP1 nor CARD8 are direct cleavage substrates. When probing DPP-9 by Western blot, you detect total protein regardless of enzymatic state. To assess activity, you would need peptidase assays or functional readouts like caspase-1 activation in DPP-9 inhibitor-treated cells.