Anti-ADAMTS-13 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAMTS-13 on Western blot?

Full-length ADAMTS-13 runs at approximately 190 kDa on reducing SDS-PAGE, though the predicted molecular weight from its 1427-amino acid sequence is closer to 154 kDa. The discrepancy reflects extensive post-translational modifications, particularly N- and O-linked glycosylation. In plasma samples or conditioned media from cells secreting ADAMTS-13, you may observe additional bands around 170-180 kDa representing partially processed forms. Under non-reducing conditions, disulfide-linked complexes can appear at higher apparent molecular weights. Always include a positive control such as human plasma or recombinant ADAMTS-13 to confirm band identity.

What dilution should I start with for Western blot and IHC with this ADAMTS-13 antibody?

For Western blot, start at 1:1000 dilution in blocking buffer as recommended, though optimal dilution depends on ADAMTS-13 expression levels in your sample. Plasma or serum typically requires 1:1000 to 1:2000; cell lysates from hepatic stellate cells or endothelial cells may need 1:500 to 1:1000. For IHC on formalin-fixed paraffin-embedded tissue, begin at 1:100 to 1:200 and titrate based on background and signal intensity. Liver and vascular endothelium serve as useful positive-control tissues. Always run a no-primary-antibody control to assess non-specific binding of your secondary reagent.

Does this antibody detect ADAMTS-13 in mouse or rat samples?

This antibody is validated for human ADAMTS-13 detection. Cross-reactivity with mouse or rat ADAMTS-13 is not validated and should be empirically tested if you plan to use rodent samples. Human and mouse ADAMTS-13 share approximately 78 percent amino acid identity, so cross-reactivity is possible but not guaranteed depending on the epitope recognized by this polyclonal. If you need to detect murine ADAMTS-13, we recommend running a test blot with mouse plasma alongside human plasma as a positive control to confirm signal before committing to larger experiments.

What positive and negative controls should I use for ADAMTS-13 Western blots?

Pooled normal human plasma diluted 1:20 to 1:50 in sample buffer serves as a reliable positive control, as ADAMTS-13 circulates constitutively at approximately 1 microgram per milliliter. Recombinant human ADAMTS-13 is another option if available. For cell lysates, hepatic stellate cells or human umbilical vein endothelial cells express ADAMTS-13 endogenously. Negative controls include plasma samples immunodepleted of ADAMTS-13 or cell lines with negligible ADAMTS-13 expression, such as most hematopoietic or epithelial lines. Plasma from patients with severe congenital ADAMTS-13 deficiency can also serve as a biological negative control.

Can I use this antibody to detect ADAMTS-13 in conditioned media from cultured cells?

Yes, this antibody detects secreted ADAMTS-13 in serum-free conditioned media from cells that express and secrete the protease. Concentrate conditioned media 10- to 20-fold using centrifugal filters with a 30 or 50 kDa molecular weight cutoff to improve detection sensitivity, especially if expression levels are low. Avoid serum-containing media for collection, as bovine serum contributes background proteins. Hepatic stellate cells and endothelial cells are the primary physiological sources. Include recombinant ADAMTS-13 or human plasma as a positive control. Detection sensitivity depends on secretion levels and culture duration; 48- to 72-hour collections generally yield better signal than shorter intervals.

Will this antibody recognize truncated or proteolytically processed forms of ADAMTS-13?

Recognition of truncated ADAMTS-13 fragments depends on the epitope location within the 1427-residue protein, which is not specified for this polyclonal antibody. Polyclonal antibodies typically recognize multiple epitopes across the immunogen, increasing the likelihood of detecting at least some processed forms. ADAMTS-13 can undergo proteolytic cleavage in vivo, generating fragments that may or may not retain the recognized epitopes. If you are studying proteolytic processing, consider testing the antibody against characterized recombinant fragments or running samples under reducing versus non-reducing conditions to identify potential fragment patterns. N-terminal versus C-terminal fragment detection may require different antibodies.

How should I store this ADAMTS-13 antibody for long-term stability?

Store the antibody at minus 20 degrees Celsius for long-term storage. Upon receipt, aliquot into working volumes to avoid repeated freeze-thaw cycles, which can reduce antibody activity and increase aggregation. For frequent use, a working aliquot can be kept at 4 degrees Celsius for up to one month in the presence of a preservative such as sodium azide, though check the product formulation details. Avoid storing diluted antibody; prepare fresh working dilutions for each experiment. If you observe increased background or decreased signal intensity over time, the antibody may have degraded; compare results against a freshly thawed aliquot.

Does ADAMTS-13 expression vary between different tissue samples for IHC?

Yes. In normal human tissues, ADAMTS-13 expression by IHC is most prominent in hepatic stellate cells within the liver and in vascular endothelial cells, particularly in smaller vessels. Expression levels are lower in most other cell types. Kidney podocytes and some stromal cells may show weak staining. When optimizing IHC protocols, use liver or vascular-rich tissues as positive controls. In disease states such as liver fibrosis or thrombotic microangiopathies, ADAMTS-13 distribution and intensity can change. Antigen retrieval is typically required for formalin-fixed paraffin-embedded sections; citrate buffer at pH 6.0 is a common starting condition.