Anti-Tripeptidyl Peptidase-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for TPP1 on a Western blot?

TPP1 is synthesised as a ~61 kDa proenzyme and undergoes proteolytic maturation in the lysosome. On Western blot, you typically observe the mature form at approximately 46 kDa, which represents the catalytically active enzyme. Depending on cell type and lysosomal processing efficiency, you may also detect the proenzyme or intermediate processing forms around 50-61 kDa. Glycosylation heterogeneity can cause band broadening. If you see multiple bands, this often reflects normal maturation rather than non-specific binding. Run a positive control lysate from a cell line with high lysosomal content to establish your expected pattern.

What starting dilution should I use for Western blot with this TPP1 antibody?

The recommended starting dilution for Western blot is 1:1000 in blocking buffer. This polyclonal antibody has been validated at this concentration for detecting endogenous TPP1 in human cell lysates. Depending on your sample TPP1 expression level, you may need to titrate between 1:500 and 1:2000. Lysosome-enriched fractions or tissues with high lysosomal content may yield stronger signal and permit higher dilution. Always include a lysate known to express TPP1 as a positive control during your initial titration to confirm antibody performance in your specific blocking and detection system.

Is this antibody validated for detecting TPP1 in species other than human?

This antibody has been validated specifically for human TPP1. Cross-reactivity with mouse or rat TPP1 is predicted based on sequence homology in the immunogen region, but has not been experimentally validated by Triple Point Biologics. If you are working with non-human samples, we recommend running a pilot blot alongside human positive control lysate to confirm detection. Sequence alignment of the epitope region against your species of interest can help predict likely reactivity, but empirical testing remains the definitive approach. Contact us if you obtain cross-species data we can add to the validation record.

Can I use this TPP1 antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are routinely validated for Western blot and immunohistochemistry as brand-wide applications. This rabbit polyclonal should be compatible with IHC on formalin-fixed paraffin-embedded tissue sections and immunofluorescence in cultured cells, though specific validation data for TPP1 in those formats are not yet available. For IF, start around 1:100-1:200 dilution; for IHC, typical working dilutions range from 1:50 to 1:200 depending on antigen retrieval method. Because TPP1 is lysosomal, expect punctate perinuclear staining. Titrate carefully and include a no-primary-antibody control to assess background in your system.

What sample preparation considerations are important for detecting lysosomal TPP1?

TPP1 is a soluble lysosomal hydrolase, so standard RIPA or NP-40 lysis buffers work well for whole-cell lysates. Include protease inhibitors to prevent degradation, though TPP1 itself is relatively stable. If you are fractionating, TPP1 partitions with the lysosome-enriched pellet in differential centrifugation but is released into the supernatant upon lysosomal lysis. Avoid prolonged incubation at acidic pH during lysis, as this may trigger autocatalytic processing or activity that complicates band interpretation. For tissue samples, fresh-frozen material generally yields cleaner results than aged or improperly stored specimens due to TPP1's role in protein catabolism.

Does TPP1 have isoforms or splice variants I should be aware of?

The TPP1 gene produces a single major transcript encoding the 563-amino-acid preproenzyme. Alternative splicing has not been widely reported for TPP1, so you should expect one principal isoform. The complexity you observe on a blot typically arises from post-translational processing rather than splice variants: the proenzyme is N-glycosylated in the ER, trafficked to lysosomes, and proteolytically cleaved to generate the mature enzyme. Loss of signal or aberrant processing patterns can occur in cells with lysosomal storage defects or trafficking mutations. If working with patient-derived samples, be aware that pathogenic TPP1 mutations underlie neuronal ceroid lipofuscinosis type 2.

How should I store this TPP1 antibody and what is the expected shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is suitable for aliquoting into 10-20 µL volumes depending on your typical usage. Polyclonal antibodies in glycerol-containing storage buffer are generally stable for at least two years at -20°C. For short-term use, a working aliquot can be kept at 4°C for up to one month. Avoid leaving the antibody at room temperature for extended periods. If you observe increased background or loss of signal over time, prepare a fresh aliquot rather than continuing with a degraded stock.

What positive control cell line or tissue should I use to validate TPP1 detection?

Most mammalian cell lines express detectable TPP1 because it is a constitutive lysosomal protease involved in housekeeping protein catabolism. HeLa, HEK293, and fibroblast lysates serve as reliable positive controls. Tissues rich in lysosomes such as liver, spleen, or kidney homogenates typically show robust TPP1 signal. If you are studying a disease model, compare your experimental sample against a wild-type or non-diseased control processed identically. For assay development, consider obtaining a characterized reference lysate or recombinant TPP1 fragment to confirm antibody specificity before moving to experimental samples with unknown expression levels.