Anti-Cathepsin G Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Cathepsin G on a Western blot?

Cathepsin G migrates at approximately 26-29 kDa on reducing SDS-PAGE, corresponding to the mature, active protease form following signal peptide and activation dipeptide cleavage. The full-length preproprotein is 255 amino acids, but the secreted mature enzyme begins at residue 27. You may occasionally observe a band near 37 kDa representing the inactive proenzyme form in certain cell lysates or granule preparations. Neutrophil and mast cell lysates typically show predominantly the 26-29 kDa species. Note that Cathepsin G is heavily glycosylated, which can cause minor MW heterogeneity between samples.

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

Start at 1:1000 dilution in blocking buffer for Western blot applications, as specified in our validation data. This dilution has been confirmed with human neutrophil lysates and provides clean signal with minimal background when using standard chemiluminescent detection. If working with samples of lower Cathepsin G abundance—such as differentiated myeloid cell lines or tissue homogenates—you may need to increase antibody concentration to 1:500. Conversely, purified enzyme or concentrated granule preparations may permit dilution to 1:2000. Always include a neutrophil lysate positive control for initial titration.

Will this antibody recognize Cathepsin G in mouse samples?

Cross-reactivity with mouse Cathepsin G is predicted but not yet validated in our laboratory. Human and mouse Cathepsin G share approximately 73% amino acid identity, with conservation highest in the catalytic domain. Researchers have reported successful detection of mouse Cathepsin G using antibodies raised against human epitopes, but signal intensity and specificity vary. We recommend validating with a characterized mouse neutrophil lysate and including appropriate knockout or depletion controls if working with mouse samples. Validated human reactivity remains the only species we confirm from primary data.

Does Cathepsin G run as a single band or should I expect multiple forms?

In neutrophil lysates, you typically observe a predominant band at 26-29 kDa representing mature Cathepsin G. Additional bands may appear depending on sample type and preparation. A higher MW band around 37 kDa corresponds to the proenzyme, more abundant in biosynthetically active cells or when protease inhibitors are present during lysis. Degradation products below 26 kDa occasionally appear if samples undergo freeze-thaw cycles or protracted handling. Cathepsin G also undergoes N-glycosylation, contributing to minor band heterogeneity. For cleanest results, lyse cells in the presence of broad-spectrum protease inhibitors and process samples promptly.

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

Human peripheral blood neutrophils or differentiated HL-60 or PLB-985 cells serve as strong positive controls; Cathepsin G constitutes approximately 2-5% of total neutrophil protein. Mast cell lines (HMC-1, LAD2) also express Cathepsin G, though at lower levels. For negative controls, use lymphocyte lysates, fibroblasts, or epithelial cell lines that lack myeloid differentiation. Recombinant Cathepsin G provides an additional positive control and confirms expected MW. Pre-incubating antibody with blocking peptide—if available—offers a specificity control. Always include a no-primary-antibody lane to assess secondary antibody cross-reactivity in complex lysates.

Can this antibody detect Cathepsin G in immunohistochemistry or immunofluorescence?

Triple Point Biologics antibodies in the protease portfolio are validated for Western blot and immunohistochemistry applications. While we have not generated immunofluorescence data specifically for this Cathepsin G antibody, polyclonal rabbit antibodies in our catalog have been successfully used for IF by customers. For IHC, antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval is a reasonable starting method for formalin-fixed paraffin-embedded tissue containing neutrophils or mast cells. Optimal dilutions for IHC generally range from 1:100 to 1:500, though titration against your specific tissue type is necessary.

How should I store this antibody and what is the expected stability?

Store the antibody at -20°C in working aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied at 1 mg/mL in PBS with carrier protein and preservative. Under these conditions, stability typically exceeds two years at -20°C. For frequent use, prepare small aliquots (10-20 µL) to minimize freeze-thaw exposure, or maintain a working dilution at 4°C with 0.02% sodium azide for up to one month. Avoid prolonged storage in frost-free freezers due to temperature cycling. Centrifuge briefly before use if any precipitate forms.

What sample preparation considerations are important for detecting Cathepsin G?

Cathepsin G is a serine protease with both auto-catalytic and trans-proteolytic activity, so include protease inhibitors (PMSF, aprotinin, or a cocktail containing serine protease inhibitors) during cell lysis. Neutrophils and mast cells contain abundant proteases that remain active post-lysis and can degrade both Cathepsin G and other proteins. Use fresh lysates when possible; if freezing is necessary, snap-freeze in liquid nitrogen and store at -80°C. RIPA or standard NP-40 lysis buffers work well. Avoid boiling samples excessively, as Cathepsin G can aggregate. Reducing conditions are standard; DTT or β-mercaptoethanol at typical concentrations are appropriate.