Anti-Elafin Rabbit Polyclonal Antibody

Frequently Asked Questions

What is the expected molecular weight for Elafin on Western blot?

Elafin migrates at approximately 12–14 kDa on reducing SDS-PAGE gels, which corresponds to its 117-amino acid secreted form. The predicted molecular weight from sequence is around 10 kDa, but the protein often runs slightly higher due to its compact, disulfide-rich structure and post-translational modifications. You may occasionally observe higher-molecular-weight species representing precursor forms or dimers, particularly in lysates from epithelial tissues where Elafin is highly expressed. Starting dilution of 1:1000 should resolve the main band in human keratinocyte or bronchial epithelial lysates under reducing conditions.

Which sample types and lysis buffers work best for detecting Elafin?

Elafin is a secreted protein enriched in epithelial tissues, so conditioned media from keratinocytes, bronchial epithelial cells, or saliva are ideal sample types. For cell lysates, RIPA buffer with protease inhibitors works well. Because Elafin is small and cysteine-rich with four disulfide bonds, reducing conditions (DTT or β-mercaptoethanol) are essential for Western blot to unfold the compact WAP domain. For immunofluorescence or IHC, standard formaldehyde fixation preserves epithelial localization. Avoid freeze-thaw cycles for conditioned media samples, as Elafin stability can decline with repeated thawing.

Does this antibody cross-react with monkey or pig Elafin?

The antibody is validated in human samples and shows predicted cross-reactivity with monkey and pig Elafin based on sequence homology in the immunogen region. Monkey PI3 shares greater than 90 percent identity with human Elafin, so cross-reactivity is likely robust. Pig Elafin has moderate homology but sufficient conservation in key epitopes. However, these reactivities remain predicted rather than experimentally confirmed. If you are working with non-human primate or porcine samples, we recommend titrating the antibody starting at 1:1000 and including a human positive control lysate to benchmark signal intensity and specificity.

What positive control tissue or cell line should I use for Elafin Western blots?

Primary human keratinocytes, bronchial epithelial cells, or immortalized lines such as HaCaT keratinocytes serve as strong positive controls because Elafin is constitutively expressed in stratified and pseudostratified epithelia. For inducible expression, treat keratinocytes or airway epithelial cells with pro-inflammatory cytokines such as IL-1β or TNF-α, which upregulate PI3 transcription. Conditioned media from these stimulated cultures often yield cleaner bands than whole-cell lysates. Negative controls include non-epithelial lines like HEK293 or Jurkat cells, which express little to no endogenous Elafin under standard culture conditions.

Can this Elafin antibody detect the protein in immunohistochemistry or immunofluorescence?

Yes, this antibody is validated for Western blot; IHC validation is in progress and immunofluorescence in addition to Western blot. In human skin biopsies, expect prominent signal in the granular and spinous layers of the epidermis, where Elafin is secreted by differentiating keratinocytes. Antigen retrieval with citrate buffer, pH 6, typically improves epitope accessibility in paraffin sections. For immunofluorescence on cultured epithelial cells, use standard formaldehyde fixation and permeabilization with Triton X-100. Elafin localizes to the cytoplasm and is often visible in vesicles prior to secretion. Starting dilution for IHC is typically 1:100 to 1:200.

Are there known isoforms or cleavage products of Elafin I should be aware of?

Elafin is synthesized as a 117-amino acid secreted protein without common splice isoforms. However, the PI3 gene also encodes a longer precursor called trappin-2, which contains an N-terminal cementoin domain that anchors Elafin to extracellular matrix via transglutaminase cross-linking. Trappin-2 runs at approximately 14–16 kDa. Depending on the immunogen region of this antibody, you may detect both forms. Additionally, elastase can cleave Elafin at specific sites during prolonged inflammation, generating smaller fragments. If you observe multiple bands, consider whether your sample contains active proteases and include protease inhibitors during lysis.

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

Store the antibody at –20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100-microgram pack size is sufficient for approximately 100 Western blots at 1:1000 dilution. Once thawed, an aliquot can be kept at 4°C with sodium azide for up to one month, though we recommend shorter-term storage for maximum sensitivity. Rabbit polyclonals from Triple Point Biologics typically remain stable for at least two years from date of manufacture when stored properly. Check the datasheet for the specific lot expiration date and avoid prolonged exposure to room temperature.

Why might I see no signal or high background when probing for Elafin?

Low or absent signal often results from using non-epithelial cell types or unstimulated cultures with low basal Elafin expression. Verify your positive control and consider cytokine stimulation to induce PI3 transcription. High background can occur if the blocking step is insufficient; rabbit polyclonals benefit from blocking with 5 percent nonfat milk or BSA for at least one hour. Elafin is a small, abundant secreted protein, so overloading gels (more than 30 micrograms total protein per lane) can cause smearing. Finally, ensure reducing conditions; the compact disulfide-bonded structure of Elafin may not unfold properly without DTT or β-mercaptoethanol.