Anti-ADAMTS-20 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-20 on a Western blot?

Full-length ADAMTS-20 has a predicted molecular weight of approximately 220 kDa based on its 1,910 amino acid sequence. However, you may observe multiple bands due to post-translational modifications, glycosylation, or proteolytic processing. The mature secreted form often runs slightly higher than predicted due to extensive glycosylation. Additionally, ADAMTS-20 is subject to cleavage by MT1-MMP and other proteases in the extracellular matrix, so processed fragments below 220 kDa are common depending on your cell or tissue lysate. Always include a recombinant control or transfected lysate to confirm band identity.

What dilution should I start with for ADAMTS-20 Western blots?

We recommend starting at 1:1,000 dilution for Western blot with this rabbit polyclonal ADAMTS-20 antibody. This dilution has been validated in our hands using human cell lysates. Depending on ADAMTS-20 expression levels in your sample, you may need to adjust between 1:500 and 1:2,000. Secreted proteins like ADAMTS-20 are often present at lower concentrations than cytoplasmic proteins, so consider concentrating conditioned media or using lysates from cells known to express ADAMTS-20. Overnight incubation at 4°C typically improves signal over 1-hour room-temperature incubation.

Will this antibody cross-react with rat or mouse ADAMTS-20?

This antibody has been validated for human ADAMTS-20 and shows predicted cross-reactivity with rat and monkey based on sequence homology in the immunogen region. We have not formally validated reactivity with mouse ADAMTS-20. Rat ADAMTS-20 shares approximately 85-90 percent sequence identity with human in the epitope region, so cross-reactivity is likely but should be confirmed empirically. If working with rodent samples, include a positive control such as transfected lysate or tissue known to express ADAMTS-20, and be prepared to optimize dilution independently from the human-validated recommendation.

Can I use this ADAMTS-20 antibody for immunofluorescence or IHC?

Yes, Triple Point Biologics antibodies are typically validated across multiple applications including immunofluorescence and immunohistochemistry, in addition to Western blot. For immunofluorescence, start with a 1:100 to 1:200 dilution and expect predominantly extracellular and extracellular matrix staining, consistent with ADAMTS-20's secreted localization. For IHC on paraffin sections, antigen retrieval is usually necessary; citrate buffer pH 6.0 is a reasonable starting point. Because ADAMTS-20 is secreted, you may observe diffuse matrix staining rather than sharp cellular localization. Include a blocking peptide or secondary-only control to confirm specificity.

What positive control should I use to validate ADAMTS-20 detection?

The most reliable positive control is a lysate or conditioned medium from cells transiently transfected with ADAMTS-20 expression plasmid. Alternatively, certain melanoma and connective tissue cell lines express detectable endogenous ADAMTS-20, though expression is often low and context-dependent. Recombinant ADAMTS-20 protein can serve as a sizing reference but may lack physiological glycosylation patterns. Avoid assuming universal expression; ADAMTS-20 is tightly regulated and not constitutively expressed in most cell types. If detection fails, confirm mRNA expression by RT-PCR before troubleshooting antibody conditions.

How should I prepare samples to preserve ADAMTS-20 for Western blot?

Because ADAMTS-20 is a secreted metalloproteinase, include protease inhibitors immediately upon sample collection. Use a cocktail containing EDTA or EGTA to inhibit metalloproteinase activity, plus serine and cysteine protease inhibitors to prevent degradation by other proteases. For conditioned media, clarify by centrifugation and either use fresh or snap-freeze aliquots; avoid repeated freeze-thaw cycles. For tissue or cell lysates, RIPA buffer with protease inhibitors works well. Keep in mind that ADAMTS-20 may remain associated with extracellular matrix components, so efficient extraction may require mechanical disruption or detergent optimization.

Does ADAMTS-20 have isoforms or splice variants I should know about?

ADAMTS-20 is primarily expressed as a single full-length isoform of 1,910 amino acids. Unlike some ADAMTS family members, alternative splicing has not been extensively characterized for ADAMTS-20. However, functional diversity arises from proteolytic processing rather than splicing. The proprotein is activated by furin-like convertases, and the mature enzyme can be further cleaved by MT1-MMP and potentially other proteases, generating N-terminal and C-terminal fragments. The C-terminal GON domain has protease-independent functions in ER-to-Golgi transport. If you observe multiple bands on Western blot, consider processing events rather than splice variants.

How should I store this ADAMTS-20 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. The antibody is supplied at 100 µg per vial; consider aliquoting into 10-20 µL volumes immediately upon receipt. For short-term use within two weeks, diluted antibody in blocking buffer with 0.02 percent sodium azide can be stored at 4°C and reused multiple times. Avoid prolonged storage of working dilutions. Under proper storage conditions at -20°C, rabbit polyclonal antibodies typically retain activity for at least two years. Do not store in frost-free freezers due to temperature cycling.