Anti-MMP-8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for MMP-8 on Western blot?

Full-length MMP-8 migrates at approximately 75 kDa, corresponding to the proenzyme form (zymogen) typically found in neutrophil granules. Upon activation by proteolytic cleavage of the N-terminal propeptide, you may observe a mature active form around 58–65 kDa. In samples from inflammatory sites or stimulated neutrophils, expect both pro- and active forms. The exact mobility depends on glycosylation state and your gel system. Recombinant MMP-8 standards often resolve near 53 kDa if the propeptide is already removed. Always include a positive control such as activated neutrophil lysate or recombinant MMP-8 to confirm band identity.

Does this antibody recognize both pro-MMP-8 and active MMP-8?

Yes, this rabbit polyclonal is raised against a large internal region (residues 46–460) that spans both the propeptide and catalytic domains, so it detects pro-MMP-8 and the activated form. Because the epitope is not solely in the propeptide that is cleaved during activation, you will capture both zymogen and mature enzyme. This is advantageous for tracking MMP-8 expression and activation status simultaneously. If you need to distinguish activation state, consider running samples under reducing conditions and comparing band patterns before and after known activators like trypsin or organomercurials in vitro.

What is the recommended starting dilution for Western blot with this MMP-8 antibody?

Start at 1:1000 in blocking buffer, which is the validated dilution for Western blot. MMP-8 is abundant in neutrophils but can be present at low levels in other cell types or tissue extracts. If signal is weak in non-neutrophil samples, try 1:500. Conversely, with purified neutrophils or PMA-stimulated cells, you may achieve cleaner blots at 1:2000. Optimal dilution depends on lysate concentration, membrane type, and detection system sensitivity. Always titrate with your specific sample; include a recombinant MMP-8 standard curve if quantification is planned.

Is this MMP-8 antibody validated for mouse and rat samples?

It is validated for human MMP-8 and predicted to cross-react with mouse, rat, non-human primate, and dog orthologs based on sequence homology in the immunogen region. Mouse MMP-8 shares approximately 78% identity with human, so cross-reactivity is likely but should be confirmed in your hands. We recommend running a positive control—such as LPS-stimulated mouse peritoneal neutrophils or rat bone marrow lysate—alongside your samples. If you observe the expected 75 kDa (pro) and 58–65 kDa (active) bands, the antibody is performing as predicted in that species.

What are suitable positive and negative controls for MMP-8 Western blots?

For positive controls, use freshly isolated human neutrophils, differentiated HL-60 cells, or recombinant human MMP-8. Stimulation with PMA, LPS, or TNF-α for 4–6 hours increases MMP-8 secretion and activation. Gingival crevicular fluid or synovial fluid from inflamed joints also contain high MMP-8 levels. Negative controls include cell lines that do not express MMP-8 (HEK293, many epithelial lines) or neutrophil lysates pre-treated with siRNA knockdown targeting MMP8. Running a lane without primary antibody helps rule out nonspecific secondary binding.

Can this antibody distinguish MMP-8 from other collagenases like MMP-1 or MMP-13?

Yes, this polyclonal is raised against a region unique to MMP-8 and does not cross-react with MMP-1 (interstitial collagenase) or MMP-13 (collagenase-3), which share functional similarity but have distinct sequences outside the conserved catalytic zinc-binding motif. MMP-8 is the predominant collagenase in neutrophils, whereas MMP-1 and MMP-13 are more common in fibroblasts, chondrocytes, and some tumor cells. If your sample contains multiple collagenases, this antibody will selectively detect MMP-8. For additional confidence, compare migration to commercial recombinant standards of each collagenase on the same blot.

How should I store this MMP-8 antibody and what is the expected shelf life?

Store the antibody at –20 °C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The lyophilized or glycerol-stabilized stock is stable for at least 12 months under these conditions. Once reconstituted or thawed, keep working aliquots at 4 °C for up to one month; add sodium azide (0.02–0.05%) as preservative if storing diluted antibody longer than a week. Do not store diluted antibody in Tris buffers containing primary amines at room temperature. Mark the date on each aliquot and monitor performance over time.

What sample preparation steps optimize MMP-8 detection in tissue lysates?

Lyse tissues in RIPA or similar buffer containing protease inhibitors—critically, omit EDTA or use a minimal concentration, as it chelates zinc and can dissociate MMP-8 from binding partners or alter epitope exposure. Include phosphatase inhibitors if studying regulated secretion. MMP-8 is secreted and often bound to extracellular matrix; brief collagenase digestion or mechanical homogenization improves recovery from fibrous tissues. Clarify lysates by centrifugation at 16,000 g for 10 minutes. Load 20–40 µg total protein per lane; neutrophil-rich samples may require less to avoid overloading.