Anti-ADAM-22 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for ADAM-22 on a Western blot?

ADAM-22 migrates at approximately 100-110 kDa on reducing SDS-PAGE, which is slightly higher than its predicted molecular weight of 99 kDa (906 amino acids). This discrepancy is likely due to post-translational modifications, particularly N-glycosylation at multiple sites within the extracellular domain. As a membrane-bound protein with a single transmembrane domain, you may observe the mature glycosylated form as the dominant band in brain lysates. If you detect multiple bands, consider that ADAM-22 may undergo differential glycosylation depending on neuronal cell type and maturation state.

What is the recommended starting dilution for ADAM-22 Western blots?

Start at 1:1000 dilution for Western blot applications, which is validated for this antibody. Because ADAM-22 expression is restricted primarily to central nervous system tissue, signal intensity will depend heavily on your sample source. Brain tissue lysates typically yield robust signal at 1:1000, but cultured neuronal cells may require optimization between 1:500 and 1:2000 depending on confluence and differentiation state. Always include a protein loading control and consider titrating the antibody with your specific lysate preparation, as detergent choice and extraction conditions affect membrane protein yield.

Does this antibody work in mouse and rat brain samples?

The antibody is validated for human samples and shows potential cross-reactivity with mouse based on sequence homology analysis. Human and mouse ADAM-22 share approximately 90% sequence identity, suggesting the epitope is likely conserved. Rat ADAM-22 has similar homology and is also predicted to cross-react. However, these species have not been formally validated in our hands, so we recommend running positive control human samples in parallel during your initial experiments and confirming band specificity by molecular weight and tissue distribution. Pan (chimpanzee) is also predicted but not validated.

What are appropriate positive and negative controls for ADAM-22 expression?

Human brain tissue lysate, particularly from cortex or hippocampus, serves as a strong positive control given ADAM-22's predominant CNS expression. Cultured primary neurons or differentiated neuroblastoma cell lines (such as SH-SY5Y after retinoic acid treatment) also express detectable levels. For negative controls, use non-neuronal tissues such as liver, lung, or HEK293 cells, which express minimal to undetectable ADAM-22. If investigating LGI1-ADAM-22 interactions, consider epilepsy-associated brain regions where this receptor complex plays a documented role in synaptic transmission. Confirm absence of signal in your negative control to verify antibody specificity.

Can I use this antibody for immunofluorescence on neuronal cultures?

This antibody is produced by Triple Point Biologics, which validates antibodies for Western blot as standard practice. Given that ADAM-22 is a cell-surface protein with an extracellular domain, it is suitable for immunofluorescence on fixed neuronal cultures. For surface staining, fix cells with 4% paraformaldehyde without permeabilization; for total ADAM-22 including intracellular pools, include a permeabilization step with 0.1-0.3% Triton X-100. Start with a 1:100-1:200 dilution and optimize based on signal-to-background ratio. ADAM-22 typically shows punctate membrane staining at synaptic sites in mature neurons.

How should I prepare neuronal samples to preserve ADAM-22 for Western blot?

ADAM-22 is a membrane-bound protein requiring detergent-based extraction. Use RIPA buffer or a comparable lysis buffer containing 1% NP-40 or Triton X-100 to solubilize membrane fractions effectively. Include protease inhibitors and work on ice to prevent degradation. For brain tissue, mechanical homogenization followed by brief sonication improves yield. Avoid repeated freeze-thaw cycles, which compromise membrane protein integrity. If signal is weak, consider enriching for membrane fractions using differential centrifugation rather than whole-cell lysates. Load 20-40 µg total protein per lane as a starting point, adjusting based on ADAM-22 abundance in your specific sample type.

Are there known ADAM-22 isoforms I should be aware of?

ADAM-22 exists primarily as a single isoform (UniProt Q9P0K1, 906 amino acids), which is the canonical form expressed in the nervous system. Unlike some ADAM family members that undergo extensive alternative splicing, ADAM-22 does not have well-characterized functional splice variants reported in the literature. You should expect a single prominent band around 100-110 kDa under reducing conditions. If you observe multiple bands, this more likely reflects differential glycosylation states or proteolytic processing rather than distinct isoforms. The protein consists of a prodomain, metalloprotease-like domain (catalytically inactive), disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domains in its full-length form.

What are the storage and handling requirements for this antibody?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase background. The 100 µg pack size is supplied as a rabbit polyclonal and typically provided in a glycerol- or BSA-containing storage buffer that remains stable for at least 12 months when stored properly. Once thawed, an aliquot can be stored at 4°C for up to one month for convenient repeated use. Always centrifuge briefly before use to pellet any aggregates that may have formed during storage. Do not store diluted antibody in working solutions; prepare fresh dilutions in blocking buffer for each experiment to maintain optimal performance.