Anti-Prolyl Oligopeptidase Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Prolyl Oligopeptidase on Western blot?

Prolyl oligopeptidase migrates as a single band at approximately 80 kDa on SDS-PAGE under reducing conditions, corresponding to its 710-amino acid sequence. The predicted molecular weight from the full-length human protein is close to this value. You should see a clean band in cytoplasmic fractions from most mammalian tissues, particularly brain, kidney, and testis where PREP is highly expressed. If you observe multiple bands, consider the possibility of proteolytic degradation during lysis; include protease inhibitors and keep samples cold throughout preparation.

What starting dilution should I use for Western blot with this PREP antibody?

Start at 1:1000 dilution for Western blot as recommended, using standard overnight incubation at 4°C in blocking buffer containing 5% non-fat milk or BSA. This polyclonal rabbit antibody typically yields strong signal from 10-20 µg total protein lysate when PREP is abundantly expressed. For low-expression samples, you may titrate up to 1:500 or increase lysate loading. Always include a positive control lysate from brain or HEK293 cells to confirm antibody performance before troubleshooting your experimental samples.

Is this Prolyl Oligopeptidase antibody validated in mouse and rat tissues?

Human reactivity is validated; mouse and rat are predicted based on sequence homology. Human PREP shares approximately 90% identity with mouse and rat orthologs, particularly in the catalytic domain, so cross-reactivity is likely. However, you should confirm performance in your species of interest by testing positive control lysates—mouse or rat brain tissue works well due to high endogenous PREP expression. If signal is weak or absent, empirical titration may be required. We classify these as potential reactivity until independently confirmed by users.

Which subcellular fraction should I use to detect PREP by Western blot?

Prolyl oligopeptidase is a cytoplasmic enzyme, so whole-cell lysates or cytoplasmic fractions will give the strongest signal. Use standard RIPA or NP-40 lysis buffers; avoid nuclear-enrichment protocols that deplete cytoplasm. If performing subcellular fractionation, verify your cytoplasmic marker (e.g., GAPDH, tubulin) to confirm enrichment. PREP is soluble and does not require detergents beyond those in typical lysis buffers. For tissues, homogenize thoroughly to release cytoplasmic contents before clarification by centrifugation at 10,000–15,000 × g.

Can I use this antibody for immunofluorescence to visualize Prolyl Oligopeptidase localization?

Triple Point Biologics antibodies are validated for Western blot; immunofluorescence performance should be empirically tested. Because PREP is cytoplasmic, you should see diffuse cytoplasmic staining if the antibody performs well in IF. Use standard methanol or 4% paraformaldehyde fixation and permeabilize with 0.1–0.3% Triton X-100. Start at 1:100–1:200 dilution and include a no-primary-antibody control to assess background. Polyclonal antibodies can sometimes show nonspecific binding in IF, so validate signal by comparing PREP-expressing versus PREP-knockdown cells if possible.

Does Prolyl Oligopeptidase have isoforms I need to account for?

Human PREP is encoded by a single gene on chromosome 6 and produces one major isoform of 710 amino acids. No functionally distinct splice variants or post-translational cleavage products are commonly reported in the literature, so you should expect a single 80-kDa band on Western blot. Polymorphisms exist in human populations, but these typically do not alter epitope recognition by polyclonal antibodies. If you observe additional bands, they more likely reflect degradation, nonspecific binding, or cross-reactivity rather than bona fide isoforms.

How should I store this Prolyl Oligopeptidase 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. For frequent use, keep a working aliquot at 4°C for up to one month; polyclonal rabbit antibodies in glycerol or similar stabilizers are generally stable under these conditions. Do not store diluted antibody; prepare fresh working dilutions in blocking buffer for each experiment. If you observe declining signal over time, prepare a fresh aliquot from the –20°C stock rather than increasing antibody concentration.

What positive and negative controls should I use when working with this PREP antibody?

Human or rodent brain lysate serves as an excellent positive control due to high endogenous PREP expression; HEK293 or HeLa lysates also work. For a negative control, consider PREP-knockout cell lines if available, or use tissues with very low expression, though few mammalian tissues entirely lack PREP. Alternatively, pre-incubate the antibody with recombinant PREP protein to competitively block binding, then compare to an unblocked sample. Include a secondary-antibody-only lane to rule out nonspecific signal from the detection system.