Anti-SPPL2A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for SPPL2A on a Western blot?

SPPL2A (UniProt Q8TCT8) runs at approximately 56-60 kDa on Western blot, consistent with the calculated molecular weight of the full-length protein. The intramembrane protease is synthesized as a single-chain polypeptide that may undergo autoproteolytic processing in some cell types, though the mature form typically migrates near the predicted size. If you observe higher-molecular-weight bands, consider that SPPL2A can form oligomeric complexes or undergo post-translational modification. Use late endosome-enriched fractions or lysates from immune cells (B cells, dendritic cells) as positive controls, since SPPL2A expression is highest in tissues involved in antigen presentation.

What dilution should I start with for Western blot using this SPPL2A antibody?

We recommend starting at 1:1000 dilution for Western blot, which has been validated for this rabbit polyclonal antibody. This dilution typically provides a strong signal from 20-30 µg of total cell lysate when SPPL2A is expressed at endogenous levels. If you are working with tissues or cell lines that express SPPL2A at low levels, you may need to increase antibody concentration to 1:500 or enrich for membrane fractions. Conversely, if using cells with high SPPL2A expression (such as activated B cells or dendritic cells), 1:2000 may suffice. Titre the antibody against your specific sample type to optimize signal-to-noise ratio.

Does this antibody cross-react with SPPL2B or other family members?

SPPL2A shares approximately 49% sequence identity with its paralog SPPL2B and lower homology with SPPL2C and SPPL3. This antibody was raised against human SPPL2A and validated for human reactivity; cross-reactivity with other SPPL family members has not been formally tested. Given the divergence between SPPL2A and SPPL2B, especially in the N-terminal and loop regions, we anticipate selectivity for SPPL2A, but if your experimental system co-expresses both isoforms, consider running lysates from SPPL2A or SPPL2B knockout cells as negative controls to confirm specificity. Alternatively, siRNA knockdown can verify band identity.

What sample preparation method works best for detecting SPPL2A in Western blot?

Because SPPL2A is an intramembrane protease embedded in late endosomal membranes, use lysis buffers containing 1% Triton X-100, NP-40, or CHAPS to efficiently solubilize membrane proteins. RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) works well for total cell lysates. Avoid boiling samples excessively; heating at 70-95°C for 5-10 minutes is sufficient and reduces aggregation of hydrophobic membrane proteins. Subcellular fractionation to enrich late endosomes or lysosomal compartments will improve signal if expression is low. Include protease inhibitors (PMSF, protease inhibitor cocktail) to prevent degradation during lysis.

Which cell lines or tissues have high endogenous SPPL2A expression for positive controls?

SPPL2A is highly expressed in immune cells, particularly B lymphocytes, dendritic cells, and monocyte-derived macrophages, where it regulates CD74 degradation and antigen presentation. The Raji B-cell line, THP-1 monocytic cells (especially after PMA differentiation), and primary human peripheral blood mononuclear cells (PBMCs) serve as reliable positive controls. Spleen, lymph node, and thymus tissue lysates also show robust SPPL2A signal. Non-immune cells typically express lower levels, so HeLa or HEK293 lysates may yield weak or absent bands unless SPPL2A is induced by interferon-gamma or other immune stimuli. Use 20-30 µg of lysate from these sources.

Can I use this SPPL2A antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are validated for Western blot, so this rabbit polyclonal is compatible with immunohistochemistry on formalin-fixed paraffin-embedded (FFPE) sections. Antigen retrieval (citrate buffer, pH 6.0, or Tris-EDTA pH 9.0) is typically required to expose epitopes in fixed tissue. For immunofluorescence on cultured cells, use methanol fixation or mild paraformaldehyde (2-4%, 10 min) followed by permeabilization with 0.1-0.5% Triton X-100, since SPPL2A resides in intracellular late endosomal compartments. Start with 1:100-1:200 dilution for IHC or IF and optimize empirically. Expect punctate perinuclear staining consistent with endosomal localization.

Is this antibody predicted to work in mouse or rat samples?

This antibody has been validated for human SPPL2A. Cross-reactivity with mouse or rat is not validated but may be predicted based on sequence homology. Human SPPL2A shares approximately 85-90% amino acid identity with mouse and rat orthologs, with conservation highest in the catalytic aspartate motifs and transmembrane domains. If you plan to use mouse or rat lysates, we recommend testing the antibody at the standard 1:1000 dilution and including a known positive control (mouse spleen or lymph node lysate). If no signal is observed, the epitope recognized by this polyclonal may lie within a divergent region. Consider species-specific alternatives if cross-reactivity is critical.

How should I store this SPPL2A 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 titre and increase aggregation. The 100 µg pack size is sufficient for approximately 100 Western blots at 1:1000 dilution. If you need to store working aliquots at 4°C for short-term use (up to one month), add sodium azide (0.02-0.05% final concentration) or store in 50% glycerol to prevent microbial growth and maintain stability. Avoid prolonged exposure to room temperature. Rabbit polyclonal antibodies typically retain activity for 12-24 months when stored properly at -20°C. Check for precipitate before use; centrifuge briefly if cloudiness appears.