Anti-ADAMTS-5 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-5 on a Western blot?

Full-length ADAMTS-5 runs at approximately 100-120 kDa on reducing SDS-PAGE, though the predicted molecular weight from the 930-amino acid sequence is around 100 kDa. You may observe additional bands corresponding to proteolytic processing products, particularly fragments retaining the catalytic domain. ADAMTS-5 undergoes furin-mediated cleavage and autocatalytic processing in vivo, generating N-terminal fragments that retain enzymatic activity. Glycosylation also contributes to the apparent molecular weight heterogeneity. If working with conditioned media or extracellular matrix preparations, expect to see processed forms alongside the full-length enzyme.

What starting dilution should I use for Western blot with this ADAMTS-5 antibody?

We recommend starting at 1:1000 dilution for Western blot applications. This dilution has been validated with human samples and typically provides clean signal with low background when using standard chemiluminescent detection. If signal is weak in your system—particularly if ADAMTS-5 expression is low or you are working with conditioned media where the protein is dilute—you can increase concentration to 1:500. Conversely, if background is high, titrate up to 1:2000. Always include a positive control lysate from chondrocytes or osteoarthritic cartilage where ADAMTS-5 is abundantly expressed.

Will this antibody work with mouse and rat samples?

Cross-reactivity with mouse and rat ADAMTS-5 is predicted based on sequence homology but has not been experimentally validated by us. Human and mouse ADAMTS-5 share approximately 89 percent sequence identity, and rat homology is similar, suggesting the antibody should recognize these orthologs. However, we recommend validating performance in your specific system before committing to large-scale experiments. Test a dilution series starting at 1:1000 and include a known positive control such as murine chondrocyte lysate or cartilage explant extract to confirm reactivity and determine optimal working concentration.

Is this antibody suitable for detecting ADAMTS-5 in cartilage tissue sections?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress and should detect ADAMTS-5 in cartilage sections. ADAMTS-5 localizes to the extracellular matrix, particularly the pericellular and territorial regions surrounding chondrocytes in articular cartilage. For IHC, antigen retrieval is typically required—heat-induced epitope retrieval in citrate buffer (pH 6.0) works well for cartilage. Expression is notably elevated in osteoarthritic cartilage compared to healthy tissue, making OA samples useful positive controls. Be aware that ADAMTS-5 may appear as diffuse extracellular staining rather than discrete cellular localization due to its secreted nature and matrix association.

What positive control should I use to validate ADAMTS-5 detection?

Human chondrocyte lysates or osteoarthritic cartilage extracts serve as excellent positive controls, as ADAMTS-5 is abundantly expressed in cartilage and upregulated in osteoarthritis. Alternatively, use lysates from cells stimulated with interleukin-1β or tumor necrosis factor-α, which induce ADAMTS-5 expression in chondrocytes and synovial fibroblasts. For extracellular experiments, concentrated conditioned medium from stimulated chondrocytes contains secreted ADAMTS-5. SW1353 chondrosarcoma cells also express detectable ADAMTS-5. If working with other tissues, skeletal muscle or aortic smooth muscle extracts may provide positive signal, as ADAMTS-5 participates in versican remodeling in these contexts.

Does this antibody recognize both pro-ADAMTS-5 and the active processed form?

The epitope specificity has not been mapped to distinguish between pro-enzyme and processed forms. Given that this is a polyclonal antibody, it likely recognizes multiple epitopes across the ADAMTS-5 protein and should detect both the zymogen and proteolytically activated forms, provided the epitopes remain intact after processing. ADAMTS-5 undergoes furin cleavage to remove the prodomain, and subsequent autocatalytic processing can generate additional fragments. If your research requires discriminating between these forms, perform Western blots under reducing conditions and examine the banding pattern—full-length pro-ADAMTS-5, mature enzyme, and processed fragments will migrate differently based on molecular weight.

Can I use this antibody to detect ADAMTS-5 in conditioned cell culture media?

Yes, ADAMTS-5 is a secreted metalloproteinase, and this antibody should detect it in conditioned media by Western blot. Concentrate the media 10- to 20-fold using centrifugal filters (30 kDa cutoff) to improve detection, as secreted levels can be low depending on cell type and culture conditions. Include protease inhibitors during collection—EDTA or 1,10-phenanthroline to inhibit metalloproteinases that might degrade ADAMTS-5. Serum-free or low-serum conditions reduce background and avoid interference from serum proteases. Load equivalent cell numbers or volumes, and include unconditioned medium as a negative control to confirm signal specificity.

How should I store this antibody and how long will it remain stable?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. Aliquot immediately upon receipt if you plan infrequent use—20 to 25 µL aliquots work well for most Western blot applications at 1:1000 dilution. The antibody remains stable for at least 12 months at -20°C when stored properly. For short-term use (up to one month), the working aliquot can be kept at 4°C with 0.02 to 0.05 percent sodium azide as preservative. Avoid storing diluted antibody; prepare working dilutions fresh in blocking buffer for each experiment.