Anti-ADAM-8 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-8 on a Western blot?

Full-length ADAM-8 runs at approximately 90-100 kDa on reducing SDS-PAGE, corresponding to the 824-residue membrane-anchored form. You may also observe lower molecular weight bands around 55-65 kDa, which likely represent proteolytically processed forms or shed ectodomain fragments. ADAM-8 undergoes post-translational modifications including glycosylation, which can broaden the band appearance. If you see multiple bands, consider that ADAM-8 is subject to autocatalytic processing and ectodomain shedding in vivo. Running a positive control lysate from activated leukocytes or monocytic cell lines will help you identify the predominant species in your experimental system.

What starting dilution should I use for Western blot with this ADAM-8 antibody?

We recommend starting at 1:1000 for Western blot, which has been validated with this polyclonal. Depending on ADAM-8 expression levels in your sample, you may need to optimize between 1:500 and 1:2000. Leukocyte-rich samples typically yield strong signal at 1:1000, while tissues with lower ADAM-8 expression may require more concentrated antibody. Use 5 percent non-fat dry milk or BSA in TBST for blocking. Because this is a rabbit polyclonal, expect good sensitivity but titre the antibody against your specific lysate type during initial experiments to establish your optimal working concentration.

Is this ADAM-8 antibody cross-reactive with mouse or rat samples?

This antibody is validated for human ADAM-8 detection. Cross-reactivity with mouse or rat has not been experimentally validated by us. Human and mouse ADAM-8 share approximately 75 percent sequence identity, so cross-reactivity is possible but not guaranteed. If you are working with rodent samples, we recommend running a pilot Western blot with mouse or rat leukocyte lysates alongside human controls. Check the band pattern at the expected molecular weight and verify specificity with a blocking peptide or siRNA knockdown if possible. For definitive rodent studies, consider a species-specific antibody.

What cell types or tissues serve as good positive controls for ADAM-8?

ADAM-8 is expressed primarily in leukocytes, particularly monocytes, macrophages, and neutrophils. THP-1 cells (human monocytic leukemia line) or PMA-activated THP-1 macrophages serve as excellent positive controls for Western blot. Peripheral blood mononuclear cells (PBMCs) isolated from whole blood also express ADAM-8. For tissue samples, spleen, lymph node, and inflamed tissue biopsies typically show detectable expression. As a negative control, consider cell lines with minimal myeloid characteristics. ADAM-8 expression is upregulated during inflammation, so stimulated or activated immune cells will give stronger signal than resting cells.

Can I use this antibody for immunofluorescence or immunohistochemistry on ADAM-8?

Our antibodies are broadly validated for immunofluorescence and immunohistochemistry applications across the catalog. While this specific ADAM-8 antibody is validated for Western blot at 1:1000, it should be compatible with IHC and IF, though optimal dilutions will differ. For IHC on paraffin sections, start with antigen retrieval using citrate buffer (pH 6.0) and test dilutions between 1:50 and 1:200. For immunofluorescence, begin around 1:100 to 1:250. Because ADAM-8 is a membrane protein, expect plasma membrane and potential cytoplasmic staining. Always include isotype controls and, if possible, a blocking peptide control to confirm specificity.

Does ADAM-8 have isoforms I should be aware of when interpreting blots?

The primary ADAM-8 isoform is the 824-amino acid full-length membrane-bound form. Alternative splicing has been reported but is not extensively characterized. The more common complexity comes from post-translational processing: ADAM-8 undergoes proteolytic maturation with removal of the prodomain, and the ectodomain can be shed from the cell surface, releasing soluble fragments. If you detect multiple bands, they likely reflect these processing events rather than distinct gene isoforms. The metalloproteinase domain remains in the larger membrane-tethered fragment. Consider whether your sample preparation captures membrane-bound, shed, or both forms depending on your experimental question.

How should I prepare cell lysates to preserve ADAM-8 for Western blot detection?

Use a RIPA or similar lysis buffer containing protease inhibitors to preserve full-length ADAM-8 and minimize artifactual degradation. Because ADAM-8 is a metalloprotease, include a broad-spectrum protease inhibitor cocktail and consider adding EDTA (5-10 mM) or a metalloprotease-specific inhibitor to prevent autocatalytic activity during lysis. Keep samples on ice and process quickly. ADAM-8 is a transmembrane protein, so ensure your lysis buffer contains sufficient detergent (1 percent Triton X-100 or NP-40) for membrane solubilization. If analyzing shed ectodomain, you can also concentrate conditioned culture medium by TCA precipitation or ultrafiltration before running alongside cell lysates.

How should I store this ADAM-8 antibody and what is the expected stability?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The antibody is supplied in a stabilizing buffer and remains stable for at least 12 months when stored properly at -20°C. For convenience during active use, a working aliquot can be kept at 4°C for up to one month, though -20°C storage is preferable for long-term retention of activity. Avoid prolonged exposure to room temperature. Do not store diluted antibody; prepare fresh working dilutions in blocking buffer for each experiment. If you observe increased background or decreased signal over time, prepare a fresh aliquot.