Anti-TLL-1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for TLL-1 on a Western blot?

TLL-1 migrates at approximately 110–120 kDa on reducing SDS-PAGE, reflecting the full-length 1,013-residue proenzyme. Because TLL-1 is a secreted metalloprotease that undergoes post-translational processing and glycosylation in the extracellular space, you may observe additional bands corresponding to maturation intermediates or proteolytic fragments, particularly in conditioned media from cells actively secreting the enzyme. If you see multiple bands, consider treating samples with PNGase F to collapse glycoforms and confirm identity by enriching secreted fractions rather than whole-cell lysates.

What dilution should I start with for Western blot detection of TLL-1?

We recommend starting at 1:1000 dilution for Western blot, which has been validated for this polyclonal antibody in human samples. Because TLL-1 is a secreted protein and may be present at low abundance in whole-cell lysates, you may need to concentrate conditioned media or enrich extracellular matrix fractions to achieve strong signal. If signal is weak, titrate between 1:500 and 1:2000 against your specific sample type. Overnight incubation at four degrees Celsius can improve sensitivity when working with low-expression systems or tissue homogenates.

Does this TLL-1 antibody cross-react with other tolloid family proteases like BMP1 or TLL-2?

Cross-reactivity with closely related astacin metalloproteases—BMP1, mammalian tolloid (mTLD), and TLL-2—has not been systematically excluded for this polyclonal reagent. These family members share conserved catalytic and CUB domains, so some cross-reactivity is possible depending on the immunogen epitope. If you need to discriminate TLL-1 from BMP1 or TLL-2 in your system, validate specificity by siRNA knockdown of TLL1 and re-probing, or compare migration patterns since BMP1 isoforms range from 90–120 kDa. Recombinant protein controls for each family member are advisable.

Can I use this antibody to detect mouse or rat TLL-1 by Western blot?

This antibody is validated for human TLL-1. Mouse and rat TLL-1 orthologs share approximately 85–90 percent sequence identity with human TLL-1, so cross-reactivity is predicted but not experimentally confirmed. If you are working with rodent samples, test the antibody at the recommended 1:1000 dilution and include a positive control such as conditioned medium from mouse embryonic fibroblasts or skeletal tissue lysate known to express Tll1. Running human recombinant TLL-1 in a parallel lane will help confirm whether any observed band corresponds to the expected ortholog.

What sample type gives the best TLL-1 signal: whole-cell lysate or conditioned medium?

TLL-1 is a secreted metalloprotease, so conditioned medium from cultured cells or extracellular matrix–enriched fractions typically yield stronger signal than whole-cell lysates. For adherent cells, collect serum-free conditioned medium after 24–48 hours, then concentrate 10- to 20-fold using centrifugal filters with a 30 kDa cutoff. If working with tissue, consider decellularized matrix preparations or sequential salt extractions to enrich extracellular proteins. Intracellular pools represent primarily immature proenzyme; if you analyze lysates, expect weaker signal unless TLL-1 is overexpressed or trafficking is impaired.

How should I store this TLL-1 antibody to maintain long-term stability?

Store the antibody at minus twenty degrees Celsius as supplied. For routine use, prepare small working aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. If you use the antibody infrequently, consider adding glycerol to a final concentration of 50 percent and storing at minus twenty degrees Celsius, or keep an undiluted aliquot at minus eighty degrees Celsius for long-term archive. Once diluted in blocking buffer, the antibody can be stored at four degrees Celsius with 0.02 percent sodium azide and re-used two to three times for Western blot applications.

What positive control should I use to confirm TLL-1 antibody specificity?

Recombinant human TLL-1 or conditioned medium from cells overexpressing TLL1 serves as a robust positive control. Developmentally relevant tissues—fetal cartilage, calvarial bone, or embryonic connective tissue lysates—endogenously express TLL-1 and provide physiological context. For validation, perform siRNA-mediated knockdown of TLL1 in a cell line that secretes the protease; loss of the 110–120 kDa band confirms specificity. Avoid using generic osteoblast or fibroblast lysates without first confirming TLL1 mRNA expression by qPCR, since basal levels can be low in standard culture conditions.

Does TLL-1 require reducing conditions on Western blot, or can I run non-reducing gels?

TLL-1 contains multiple disulfide bonds critical for folding of its CUB and EGF-like domains, but reducing SDS-PAGE is standard and recommended for Western blot detection. Under non-reducing conditions, the protein may migrate anomalously or aggregate, complicating interpretation. If you are investigating multimeric complexes or cofactor interactions in native extracellular matrix, non-reducing gels can be informative, but expect altered migration and potentially weaker signal. For routine detection and molecular-weight confirmation, always use reducing sample buffer with dithiothreitol or beta-mercaptoethanol and heat samples at 95–100 degrees Celsius for five minutes.