Anti-Signal Peptide Peptidase Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Signal Peptide Peptidase on Western blot?

Signal peptide peptidase (SPP, HM13) migrates at approximately 42 kDa on SDS-PAGE under reducing conditions, corresponding to its 377-amino acid sequence. The protein is a polytopic membrane protein with multiple transmembrane domains, so complete denaturation with adequate detergent is important for consistent migration. Some labs report a minor higher-molecular-weight species around 50 kDa, potentially reflecting post-translational modifications or incomplete denaturation. Always include a positive control such as HeLa or HEK293 lysate, where SPP is endogenously expressed at moderate levels in the ER fraction.

What is the recommended starting dilution for this SPP antibody in Western blot?

We recommend starting at 1:1000 dilution in blocking buffer (5 percent non-fat milk or BSA in TBST) for overnight incubation at 4°C. This polyclonal rabbit antibody has been validated at this dilution in human lysates. Depending on your sample type and SPP expression level, you may optimize between 1:500 and 1:2000. Membrane proteins like SPP often require stronger extraction conditions—consider RIPA buffer supplemented with 1 percent SDS or urea-based lysis for optimal solubilization from ER membranes. Titre the antibody with your specific lysate preparation to establish your working dilution.

Does Signal Peptide Peptidase have isoforms or splice variants I should know about?

The HM13 gene encoding SPP produces a single major isoform of 377 amino acids. There are four additional human SPP-like proteins (SPPL2a, SPPL2b, SPPL2c, SPPL3) that share sequence homology and intramembrane protease activity but are distinct gene products with different tissue distribution and substrate specificity. This antibody is raised against SPP (HM13) and has not been validated for cross-reactivity with SPP-like family members. If you are studying the broader SPP family, confirm specificity by siRNA knockdown or verify the expected subcellular localization, as SPP is ER-resident while SPPL proteins differ.

Will this antibody recognize mouse or rat Signal Peptide Peptidase?

This antibody is validated for human SPP. Mouse and rat SPP (HM13 orthologs) share approximately 85 percent amino acid identity with human SPP, so cross-reactivity is possible but not validated by us. If you plan to use mouse or rat samples, we recommend empirical testing at the standard 1:1000 dilution with appropriate positive controls such as mouse liver or brain lysate, where SPP is expressed. Consider running a parallel human lysate to compare signal intensity and specificity. Predicted cross-reactivity based on sequence homology does not guarantee antibody performance; always validate in your species of interest.

What positive control lysate should I use for Signal Peptide Peptidase Western blots?

HeLa, HEK293, or Jurkat whole-cell lysates are reliable positive controls, as SPP is constitutively expressed in most human cell lines due to its housekeeping role in ER-associated signal peptide cleavage. For enriched signal, prepare microsomal or ER membrane fractions, since SPP is an ER-resident intramembrane protease. Human liver or placenta tissue lysates also express SPP at detectable levels. Avoid using cytosolic fractions as negative controls unless you have confirmed clean fractionation; residual membrane contamination will produce false positives. A blocking peptide or siRNA knockdown lysate serves as a specificity control.

How should I store this Signal Peptide Peptidase antibody and what is the shelf life?

Store the antibody at –20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The 100 µg stock supplied in glycerol-containing buffer is stable for at least 12 months at –20°C. For convenience, you may prepare working dilutions in blocking buffer with 0.02 percent sodium azide and store at 4°C for up to two weeks. Do not store diluted antibody long-term. After thawing an aliquot, keep on ice during use and return promptly to –20°C. If you observe increased background or loss of signal, prepare a fresh aliquot.

Can I use this SPP antibody for immunofluorescence or immunohistochemistry?

Triple Point Biologics antibodies are validated for Western blot as standard practice. For immunofluorescence of SPP, choose a permeabilization method compatible with ER membrane proteins—0.1 percent Triton X-100 or digitonin works well. SPP localizes to the ER, so expect a reticular perimuclear staining pattern. Start at 1:100 to 1:200 dilution for IF. For IHC on paraffin sections, antigen retrieval (citrate buffer, pH 6.0, heat-induced) is typically required for membrane proteases. We have not formally validated dilutions for IF or IHC on this specific clone; empirical optimization is necessary.

What lysis buffer is best for extracting Signal Peptide Peptidase from cells?

Because SPP is a polytopic ER membrane protein with multiple transmembrane helices, standard non-denaturing buffers may incompletely solubilize it. RIPA buffer (150 mM NaCl, 1 percent NP-40, 0.5 percent deoxycholate, 0.1 percent SDS, 50 mM Tris pH 8.0) supplemented with protease inhibitors is a good starting point. For stubborn membrane proteins, increase SDS to 1 percent or add 6–8 M urea. Sonication or multiple freeze-thaw cycles can improve yield. Avoid mild non-ionic detergents alone, as they leave SPP partially membrane-associated, leading to weak or smeared Western blot signal.