Anti-ADAM-19 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-19 on a Western blot?

Full-length ADAM-19 migrates at approximately 105-110 kDa on reducing SDS-PAGE, corresponding to the mature processed form after removal of the prodomain. You may also detect higher molecular weight species around 120-130 kDa representing the proenzyme or glycosylated variants, as ADAM-19 contains multiple N-glycosylation sites. Some cell types show additional lower bands around 85-90 kDa corresponding to further proteolytic processing or alternative translation products. Band pattern depends heavily on cell type and activation state, so include a positive control lysate from ADAM-19-expressing cells such as primary osteoblasts or neuronal cultures.

What dilution should I start with for Western blot with this ADAM-19 antibody?

We recommend starting at 1:1000 dilution for Western blot based on validation data. This polyclonal antibody typically provides good signal-to-noise at this concentration with standard chemiluminescent detection. If your ADAM-19 expression is low, you can titrate up to 1:500, though this may increase background in some lysates. For IHC or immunofluorescence applications, start at 1:100 to 1:200 and optimize based on your fixation method and tissue type. Always include a no-primary control to assess non-specific binding, particularly important with metalloproteases that can be abundant in extracellular matrix-rich samples.

Does this antibody cross-react with other ADAM family members?

This rabbit polyclonal was raised against human ADAM-19 and shows validated specificity for ADAM-19 in human samples. The ADAM family shares structural homology, particularly in the metalloprotease and disintegrin domains, so cross-reactivity with closely related family members such as ADAM-9, ADAM-10, or ADAM-12 is possible but has not been systematically tested. If you are working with samples expressing multiple ADAM proteases, we recommend verifying specificity by siRNA knockdown of ADAM-19 or by running parallel blots with lysates from ADAM-19-null cells if available. Peptide competition experiments can also confirm on-target binding.

Will this antibody work for mouse or rat samples?

Human ADAM-19 shows approximately 88 percent sequence identity with mouse and 87 percent with rat orthologs, and we predict cross-reactivity based on conserved epitope regions. However, we have not formally validated this antibody against mouse or rat lysates. If you plan to use rodent samples, we recommend testing at the same 1:1000 starting dilution with appropriate positive controls such as mouse primary calvarial osteoblasts or rat brain lysate, both of which express endogenous ADAM-19. Run a human lysate in parallel to confirm that band migration and intensity are comparable between species.

What sample preparation considerations are important for detecting ADAM-19?

ADAM-19 is a transmembrane metalloprotease that undergoes autocatalytic processing, so sample preparation timing matters. Lyse cells quickly in the presence of protease inhibitors to minimize artifactual processing; include EDTA or a metalloprotease inhibitor cocktail to prevent further autocatalytic cleavage during lysis. Because ADAM-19 is membrane-associated, use RIPA or a detergent-containing buffer rather than mild lysis conditions. Avoid freeze-thaw cycles, which can alter the proenzyme-to-mature enzyme ratio. For IHC, formalin fixation works well; antigen retrieval with citrate buffer pH 6.0 typically improves signal in paraffin-embedded sections.

What positive control should I use to confirm ADAM-19 detection?

ADAM-19 is expressed at moderate to high levels in primary osteoblasts, skeletal muscle, and brain tissue, particularly in regions undergoing active neuregulin signaling. For Western blot, human osteosarcoma lines such as MG-63 or Saos-2 provide reliable positive controls. Mouse or rat primary calvarial osteoblasts also express ADAM-19 strongly during differentiation. For neuronal applications, brain lysates or differentiated neuroblastoma cells work well. If endogenous expression is too low in your experimental system, consider transiently transfecting a full-length ADAM-19 expression construct as a sizing and specificity control on the same blot.

Can this antibody distinguish between the proenzyme and mature active form of ADAM-19?

This polyclonal antibody recognizes epitopes present in both the proenzyme and mature forms, so it will detect both species if present in your sample. You will typically see the mature form around 105-110 kDa and may see the proenzyme at higher molecular weight. The relative ratio depends on cellular activation state and autocatalytic processing efficiency. To specifically track activation, run samples under reducing conditions and compare band patterns between resting and stimulated cells. If you need to selectively detect only the active enzyme, you would require an antibody against the neoepitope generated after prodomain cleavage, which is not the design of this reagent.

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

Store the antibody at -20°C in the supplied glycerol-containing buffer. Under these conditions, the antibody remains stable for at least 12 months from the date of receipt. Avoid repeated freeze-thaw cycles; for frequent use, consider making single-use aliquots. If you observe reduced signal over time, check for precipitation or turbidity, which can occur if the vial has been temperature-cycled. Do not store diluted antibody in working solution for more than one week at 4°C; prepare fresh dilutions for best results. Adding 0.02 percent sodium azide to working dilutions can extend stability if you need to reuse a diluted stock.