Anti-Granzyme K Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Granzyme-K on a Western blot?

Human Granzyme-K (GZMK) migrates at approximately 28-30 kDa on reducing SDS-PAGE, corresponding to the mature secreted form after signal peptide cleavage. The full-length protein including the signal sequence is 262 amino acids, but the active enzyme is processed to roughly 245 residues upon secretion by cytotoxic lymphocytes. You may occasionally observe higher molecular weight bands around 60 kDa representing dimers or glycosylated forms, though GZMK is not heavily glycosylated. If your lysate contains high protease activity, consider including protease inhibitors during extraction to prevent degradation artifacts below 28 kDa.

Which cell types express Granzyme-K and what positive controls should I use?

GZMK is expressed predominantly in activated CD8+ cytotoxic T lymphocytes and natural killer cells, with lower levels in certain CD4+ T-cell subsets. For Western blot or IHC positive controls, use lysates from stimulated human PBMCs, purified NK cells, or CD8+ T-cells activated with anti-CD3/CD28 or PMA/ionomycin for 24-72 hours. Resting lymphocytes express minimal GZMK, so activation is typically required. Cell lines such as NK-92 or YT can serve as convenient positive controls. Negative controls include non-lymphoid tissues like HeLa or HEK293 cells, which do not express detectable GZMK under standard culture conditions.

What starting dilution should I use for Western blot with this Granzyme-K antibody?

We recommend starting at 1:1000 dilution in blocking buffer for Western blot, which has been validated with this rabbit polyclonal antibody. Depending on GZMK expression levels in your sample and your detection system sensitivity, you may optimize between 1:500 and 1:2000. Use 20-50 µg total protein from lymphocyte lysates per lane. Because GZMK is a secreted protein, consider probing both cell lysates and culture supernatants or serum samples. For supernatants, you may need to concentrate samples by TCA precipitation or ultrafiltration and use a more concentrated antibody dilution around 1:500 to detect secreted enzyme.

Does this Granzyme-K antibody cross-react with other granzyme family members?

This antibody is raised against human Granzyme-K and has been validated for specificity to GZMK. Given the sequence diversity among granzyme family members (Granzymes A, B, H, K, and M share limited homology outside their catalytic triads), cross-reactivity with other granzymes is unlikely but has not been exhaustively tested against all paralogs. If you are working with samples expressing multiple granzymes, such as activated CTLs that co-express Granzyme B, we recommend including knockout or knockdown controls where feasible. The predicted molecular weights differ sufficiently (GZMK ~28 kDa versus GZMB ~27 kDa, GZMA ~29 kDa) that cross-reactivity may be difficult to exclude by size alone on Western blots.

Will this antibody work with mouse or rat samples for Granzyme-K detection?

This antibody has been validated for human GZMK and reactivity with mouse or rat orthologs has not been confirmed. Human GZMK shares approximately 70-75 percent sequence identity with mouse Gzmk, so cross-reactivity is theoretically possible but cannot be guaranteed without empirical testing. If you require detection in rodent samples, we suggest testing the antibody at the recommended 1:1000 dilution alongside a known human positive control. Alternatively, consider species-specific antibodies raised against mouse or rat Gzmk peptides. Predicted cross-reactivity based on epitope homology is not a substitute for experimental validation in your specific system.

Can this antibody detect extracellular or secreted Granzyme-K in biological fluids?

Yes, because GZMK is a secreted serine protease released by degranulation from cytotoxic lymphocytes, this antibody can detect extracellular GZMK in culture supernatants, serum, or plasma. Concentrations in biological fluids are typically lower than intracellular levels, so you may need to concentrate samples before Western blot or use more sensitive methods like ELISA. For serum or plasma, collect samples with protease inhibitors to prevent GZMK autodegradation or cleavage by other proteases. GZMK binds heparan sulfate glycosaminoglycans, so it may associate with extracellular matrix or cell surfaces; consider this when interpreting immunofluorescence or IHC staining patterns in tissue sections.

How should I store this Granzyme-K 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 antibody is supplied at 100 µg and is stable for at least 12 months from the date of receipt when stored properly. For short-term use (up to one month), an aliquot can be kept at 4°C with 0.02-0.05 percent sodium azide as preservative. Do not store diluted antibody in working solutions for extended periods; prepare fresh dilutions for each experiment. If you observe increased background or reduced signal over time, it likely indicates antibody degradation and a fresh aliquot should be thawed.

What sample preparation considerations are important for detecting Granzyme-K in T-cell lysates?

Because GZMK is stored in cytotoxic granules and secreted upon T-cell activation, sample preparation depends on whether you want to measure intracellular stores or secreted enzyme. For total cellular GZMK, lyse cells immediately in RIPA or similar buffer containing protease inhibitors (avoid serine protease inhibitors like PMSF at high concentration, which may interfere if you later assay GZMK activity, though they are fine for Western blot). Activated T-cells secrete GZMK rapidly, so if measuring intracellular pools, minimize delays between stimulation and lysis. For secreted GZMK, collect supernatants at defined time points post-activation and clarify by centrifugation to remove cells and debris before concentration or direct analysis.