Anti-MMP-25 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for MMP-25 on a Western blot?

MMP-25 migrates at approximately 63 kDa on reducing SDS-PAGE, corresponding to the full-length 562-amino acid protein including its signal peptide, prodomain, catalytic domain, hinge region, and transmembrane domain. Some labs observe additional bands around 54 kDa representing the activated form after prodomain cleavage. Because MMP-25 is a membrane-anchored proteinase, you may also detect high-molecular-weight species if the protein remains in oligomeric complexes or associated with other membrane proteins. Glycosylation can shift the apparent mass slightly depending on cell type and post-translational modification status.

What is the recommended starting dilution for MMP-25 antibody in Western blot?

We recommend starting at 1:1000 dilution in blocking buffer for Western blot applications with this rabbit polyclonal MMP-25 antibody. This dilution has been validated with human lysates. Depending on MMP-25 expression levels in your sample, you may need to titrate between 1:500 and 1:2000 to optimize signal and minimize background. Leukocyte and activated neutrophil lysates typically yield robust signal at 1:1000, whereas samples from tissues with lower endogenous MMP-25 expression may require a more concentrated antibody. Include a positive control lysate from peripheral blood leukocytes to benchmark sensitivity in your system.

Is this MMP-25 antibody validated for monkey or mouse samples?

This antibody is validated for human MMP-25 and shows predicted cross-reactivity with monkey (non-human primate) samples based on sequence homology in the immunogen region. We have not formally validated reactivity in mouse, rat, or other rodent species. Human and cynomolgus monkey MMP-25 share high sequence identity in the catalytic and hinge domains, so the antibody should recognize primate orthologs. If you are working with mouse samples, we recommend running a positive human control in parallel and verifying any bands by siRNA knockdown or peptide competition, as cross-species reactivity is not guaranteed.

What sample preparation works best for detecting membrane-bound MMP-25?

Because MMP-25 is a membrane-type MMP with a transmembrane domain, use lysis buffers containing non-ionic detergents such as 1% Triton X-100, 1% NP-40, or RIPA buffer to solubilize membrane proteins effectively. Avoid harsh ionic detergents like SDS during lysis, as they can denature the epitope. For Western blot, load 20–50 µg total protein per lane; leukocyte-enriched samples or activated neutrophil lysates provide the best positive controls given MMP-25's restricted tissue distribution. If signal is weak in whole-cell lysates, consider enriching the membrane fraction by ultracentrifugation to concentrate MMP-25 away from cytosolic contaminants.

Does MMP-25 have splice isoforms I need to account for?

MMP-25 is encoded by a single gene on chromosome 16 and does not have well-characterized splice isoforms reported in major databases. The canonical 562-amino acid isoform is the predominant form expressed in leukocytes. You may observe proteolytic processing variants on Western blot: the inactive zymogen (~63 kDa) and the activated form after furin-mediated or autocatalytic cleavage of the prodomain (~54 kDa). Unlike some other MMPs with secreted and membrane-bound splice variants, MMP-25 remains anchored to the membrane via its C-terminal transmembrane domain in all reported functional studies, so focus on membrane-enriched fractions for optimal detection.

Can I use this antibody for immunohistochemistry on human tissue sections?

Yes, Triple Point Biologics antibodies are validated for immunohistochemistry applications, and this rabbit polyclonal should work on formalin-fixed paraffin-embedded or frozen human tissue sections. For IHC, start with a 1:100 to 1:200 dilution and perform antigen retrieval using citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0) for FFPE sections to unmask epitopes. MMP-25 expression is highest in leukocyte-rich tissues such as spleen, lymph node, and inflamed tissues with neutrophil infiltration. Include a known MMP-25-positive tissue control and a no-primary-antibody negative control to validate staining specificity in your hands.

What are the best positive and negative controls for MMP-25 experiments?

For positive controls, use lysates from peripheral blood leukocytes, activated human neutrophils, or differentiated myeloid cell lines such as HL-60 or THP-1 cells treated with PMA to induce MMP-25 expression. These cell types exhibit robust endogenous MMP-25 levels. Negative controls should include tissues or cell lines with minimal MMP-25 expression, such as most epithelial cell lines or fibroblasts. For antibody specificity controls, consider siRNA knockdown of MMP-25 in a positive cell line or peptide competition with the immunogen if available. Running samples with and without reducing agent can also help confirm the monomeric membrane-bound form.

How should I store the MMP-25 antibody and what is its 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 dividing into 10–20 µL working aliquots upon receipt. Once thawed, an aliquot can be kept at 4°C for up to one month if supplemented with 0.02% sodium azide as a preservative, though we recommend single-use aliquots for most consistent results. Typical shelf life is two years from manufacture date when stored properly at –20°C. Avoid prolonged exposure to room temperature and never refreeze a thawed aliquot multiple times.