Anti-Thimet Oligopeptidase Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Thimet Oligopeptidase on a Western blot?

Thimet oligopeptidase (THOP1) migrates at approximately 78 kDa on SDS-PAGE, consistent with its 689-amino acid sequence and historical designation as MP78. You should see a single prominent band in most cell lysates. The predicted molecular weight from UniProt P52888 is close to this observed size. If you detect higher-molecular-weight species, consider that THOP1 can exist in oligomeric complexes under certain conditions, though monomeric enzyme is predominant in reducing sample buffer. Brain and neuronal lysates typically yield the strongest signal given THOP1's role in neuropeptide metabolism.

Does this Thimet Oligopeptidase antibody cross-react with mouse and rat samples?

This polyclonal antibody is validated for human THOP1 and predicted to recognize mouse, rat, monkey, dog, and pig orthologues based on epitope sequence conservation. Human THOP1 shares approximately 90 percent identity with rodent orthologues across the immunogen region, making cross-reactivity likely. However, we recommend empirical titration when moving to a new species. Start at the 1:1000 dilution validated for human samples and adjust as needed. Mouse and rat brain lysates are good positive controls since THOP1 is abundantly expressed in nervous tissue across mammals.

What starting dilution should I use for Western blot with this THOP1 antibody?

Begin at 1:1000 in your blocking buffer for Western blot, the dilution at which this antibody has been validated against human samples. Incubate overnight at four degrees Celsius for optimal signal. If background is low and you need stronger signal, you can concentrate to 1:500. Conversely, if you see high background, dilute to 1:2000. THOP1 is a cytoplasmic enzyme moderately expressed in most tissues, so 20–40 micrograms of total cell lysate per lane is a reasonable loading range. Brain, testis, and certain cancer cell lines show higher endogenous levels.

Can I use this antibody for immunofluorescence or immunohistochemistry?

Yes, Triple Point Biologics antibodies are validated for immunofluorescence and immunohistochemistry in addition to Western blot. For IF, start at 1:100 to 1:200 dilution. THOP1 localizes primarily to the cytoplasm, so you should observe diffuse cytoplasmic staining in positive cells. For IHC on paraffin sections, antigen retrieval in citrate buffer (pH 6.0) is recommended. Neuronal tissues, particularly cortex and hippocampus, serve as excellent positive controls given THOP1's established role in neuropeptide degradation. Optimize dilution empirically, as fixation and permeabilization protocols influence epitope accessibility.

What are good positive and negative controls for Thimet Oligopeptidase expression?

Brain lysate—particularly from cortex, hippocampus, or hypothalamus—serves as a robust positive control because THOP1 is highly expressed in neurons for neuropeptide metabolism. Testis also shows strong expression. For cell lines, SH-SY5Y neuroblastoma or HEK293 cells are reliable positives. As a negative control, consider THOP1 knockdown or knockout lysates if available. Alternatively, pre-incubate the antibody with recombinant THOP1 or the immunizing peptide to demonstrate specificity by competition. Include a no-primary-antibody lane to assess non-specific binding of your secondary reagent.

Does Thimet Oligopeptidase have multiple isoforms I should be aware of?

Thimet oligopeptidase is encoded by a single gene (THOP1, UniProt P52888) and the canonical 689-amino acid isoform is predominant in most tissues. Alternative splicing has not been extensively reported, so you should expect one major band at approximately 78 kDa. The enzyme's activity depends on its zinc metallopeptidase domain; inactive mutants or degradation products may appear at lower molecular weights if samples are improperly handled. Some studies describe mitochondrial or nuclear localization under specific conditions, but cytoplasmic localization is the norm. If you detect multiple bands, verify sample integrity and protease inhibitor efficacy.

How should I prepare cell lysates to preserve Thimet Oligopeptidase for Western blot?

Lyse cells in RIPA or a similar detergent buffer supplemented with protease inhibitors, including metalloprotease inhibitors if you are studying THOP1 substrates simultaneously. THOP1 is a zinc metallopeptidase but is stable in standard lysis buffers. Keep samples on ice and process quickly; THOP1 itself is stable, but it can degrade bioactive peptides in your lysate if you delay addition of inhibitors. Avoid repeated freeze-thaw cycles, which can cause aggregation or precipitation. Boil samples in reducing Laemmli buffer for five minutes before loading to ensure complete denaturation and optimal band resolution at 78 kDa.

How should I store this Thimet Oligopeptidase antibody and what is the shelf life?

Store the antibody at minus twenty degrees Celsius in single-use aliquots to avoid repeated freeze-thaw cycles, which can compromise antibody integrity. The 100-microgram pack size allows you to prepare ten 10-microgram aliquots if you run occasional blots. For short-term use (up to one month), you may keep a working aliquot at four degrees Celsius in the presence of 0.02 percent sodium azide or similar preservative. Triple Point Biologics has validated this polyclonal under standard storage conditions; expect stable performance for at least one year when stored properly. Always centrifuge briefly before use to pellet any precipitate.