Anti-ADAMTS-4 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-4 on Western blot?

Full-length ADAMTS-4 (837 amino acids, UniProt O75173) has a calculated molecular weight of approximately 100 kDa. On reducing SDS-PAGE you will typically observe the mature protein around 68-75 kDa after cleavage of the signal peptide and pro-domain. ADAMTS-4 undergoes further autocatalytic processing in the extracellular space, so depending on your sample source you may detect multiple bands including N-terminal fragments around 53 kDa. Conditioned media from chondrocytes or cartilage explants often show these processed forms more prominently than whole-tissue lysates.

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

We recommend starting at 1:1000 dilution in blocking buffer for Western blot. This polyclonal antibody has been validated at this dilution in human samples. Optimal dilution may vary with your detection system, transfer efficiency, and ADAMTS-4 expression level in your sample. Cartilage extracts and conditioned media from IL-1β-stimulated chondrocytes typically express ADAMTS-4 at levels detectable at 1:1000 to 1:2000. For low-expression samples such as unstimulated fibroblasts, you may need to concentrate conditioned media or test dilutions down to 1:500.

Does this antibody cross-react with mouse and rat ADAMTS-4?

Human ADAMTS-4 is validated; mouse and rat are predicted cross-reactive based on sequence homology. Human and mouse ADAMTS-4 share approximately 88 percent amino acid identity, with high conservation in the metalloproteinase and disintegrin-like domains. Rat is similarly conserved. We have not formally validated this antibody in rodent samples, so if you are working with mouse or rat cartilage lysates, plan to test the recommended 1:1000 dilution and include a positive control from human chondrocyte or synovial fibroblast lysate for comparison.

What sample preparation is best for detecting secreted ADAMTS-4?

ADAMTS-4 is a secreted proteinase localized to the extracellular matrix, so conditioned serum-free media collected after 24-48 hours is often the richest source. Concentrate media 10- to 20-fold using centrifugal filters (10 kDa cutoff) to improve detection. For tissue samples, extract cartilage or connective tissue in buffer containing protease inhibitors (EDTA-free if you plan enzymatic assays) and include a brief centrifugation to remove insoluble collagen. Avoid repeated freeze-thaw cycles, which can promote autocatalytic cleavage. Include a loading control such as total protein stain, since traditional housekeepers are absent in serum-free media.

What are appropriate positive and negative controls for ADAMTS-4 experiments?

Human chondrocytes or synovial fibroblasts stimulated with IL-1β (10 ng/mL) or TNF-α for 24 hours serve as reliable positive controls, since these cytokines strongly induce ADAMTS-4 expression. Osteoarthritic cartilage explants also express high levels. For a negative control, use unstimulated chondrocytes or tissues with negligible ADAMTS-4 expression, such as liver or kidney lysates. If testing aggrecanase activity, consider recombinant ADAMTS-4 as a specificity control. Omission of primary antibody or pre-incubation with blocking peptide (if available) provides additional confirmation of signal specificity.

Can I use this antibody for immunohistochemistry on cartilage sections?

Triple Point Biologics antibodies are validated for Western blot; IHC application data is in progress, and ADAMTS-4 localization in cartilage extracellular matrix is a common use case. For paraffin-embedded sections, antigen retrieval is typically required; citrate buffer (pH 6.0) heated retrieval works well for ADAMTS-4. Because the protein is secreted and matrix-associated, expect pericellular and territorial staining around chondrocytes, with stronger signal in degraded or osteoarthritic cartilage. Optimal antibody dilution for IHC is often 2- to 5-fold more concentrated than Western blot; start around 1:200 to 1:500 and titrate.

Will this antibody distinguish between active and pro-form ADAMTS-4?

This polyclonal antibody recognizes epitopes present in both the pro-enzyme and mature ADAMTS-4, so it does not selectively detect only the active form. ADAMTS-4 is synthesized as a zymogen and activated by furin-like proprotein convertases that cleave the pro-domain. On Western blot you can infer activation status by molecular weight—full-length pro-form runs higher than the processed active enzyme—but the antibody itself binds both. If you need to specifically measure active ADAMTS-4, consider an aggrecanase activity assay or an antibody raised against a neo-epitope generated after pro-domain removal.

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

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The 100 µg format is supplied as a liquid in phosphate-buffered saline with preservative. Once thawed, an aliquot remains stable at 4°C for up to one month for routine use; keep working aliquots separate from long-term stock. Do not freeze diluted antibody in blocking buffer, as carrier proteins compromise stability. Under these conditions, shelf life is typically two years from date of manufacture. Check the product datasheet or vial label for the specific expiration date.