Anti-BACE-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for BACE-2 on Western blot?

Full-length BACE-2 migrates at approximately 56-60 kDa on reducing SDS-PAGE, reflecting the 518-amino acid precursor with post-translational modifications including N-glycosylation. You may observe additional bands around 45-50 kDa corresponding to the mature, processed form after signal peptide cleavage. Because BACE-2 is a type I transmembrane protein with multiple glycosylation sites, apparent molecular weight can vary depending on cell type and glycosylation state. Treatment with PNGase F or other deglycosylating enzymes will shift the band to approximately 47 kDa, confirming antibody specificity if band identity is uncertain.

How do I distinguish BACE-2 from BACE-1 in my samples?

BACE-1 and BACE-2 share approximately 64% sequence homology and both cleave APP, but they differ in tissue distribution and molecular weight. BACE-1 runs at roughly 70 kDa, while BACE-2 appears at 56-60 kDa, allowing separation by Western blot. This antibody was raised against BACE-2-specific epitopes and does not cross-react with BACE-1 in validated human samples. If working with neuronal lysates where BACE-1 is abundant, running a BACE-1 positive control in parallel confirms you are detecting the correct paralog. BACE-2 predominates in non-neuronal tissues including pancreas, kidney, and melanocytes.

What is the recommended starting dilution for Western blot with this BACE-2 antibody?

Start at 1:1000 dilution in 5% non-fat dry milk or BSA in TBST for overnight incubation at 4°C. This dilution has been validated for detection of endogenous BACE-2 in human cell lysates. If signal is weak in your specific system, you can increase antibody concentration to 1:500, though this may increase background. For overexpressed BACE-2, 1:2000 often suffices. Because BACE-2 is a low-to-moderate abundance membrane protein in most cell types, load at least 30-50 µg total protein per lane and consider enriching membrane fractions for optimal sensitivity.

Which cell lines serve as good positive controls for BACE-2 expression?

Human pancreatic beta cell lines such as EndoC-βH1 or MIN6 express detectable endogenous BACE-2 and serve as reliable positive controls. Melanoma cell lines including SK-MEL-28 also express BACE-2 due to its role in PMEL processing during melanogenesis. HEK293T cells transfected with BACE-2 expression plasmid provide a strong positive control for antibody validation. For negative controls, neuronal cell lines such as SH-SY5Y express predominantly BACE-1 with minimal BACE-2, allowing you to confirm antibody specificity. Kidney-derived cell lines such as HK-2 show moderate BACE-2 expression and represent physiologically relevant models.

Is this BACE-2 antibody validated for mouse and rat samples?

Human reactivity is validated; mouse and rat are predicted based on sequence homology. Human and mouse BACE-2 share approximately 87% identity across the immunogen region, and human-rat homology is similarly high, suggesting cross-reactivity is likely. However, we have not empirically tested rodent lysates with this lot. If working with mouse or rat samples, plan to validate using a known BACE-2-expressing tissue such as pancreas or kidney, and include a species-matched positive control. Optimal dilution may require titration. Predicted cross-reactivity to non-human primate, canine, and porcine BACE-2 is based on sequence alignment but remains unverified.

Can I use this antibody for immunofluorescence of BACE-2 in fixed cells?

Yes, this antibody is validated for Western blot; immunofluorescence validation is in progress in addition to Western blot. For fixed cells, start with 1:100 to 1:200 dilution in blocking buffer after permeabilization with 0.1-0.3% Triton X-100. BACE-2 localizes primarily to the plasma membrane and intracellular vesicular compartments including endosomes and the Golgi. Methanol fixation at -20°C for 10 minutes often yields cleaner membrane staining than paraformaldehyde, though PFA fixation (4%, 15 minutes) is also compatible. Include a secondary-only control to assess non-specific binding, and consider co-staining with a membrane or organelle marker to confirm localization.

What sample preparation is best for detecting endogenous BACE-2 by Western blot?

Because BACE-2 is a transmembrane protein, membrane-enriched fractions yield stronger signal than whole-cell lysates. Use RIPA buffer with protease inhibitors for whole-cell lysis, or fractionate using a membrane protein extraction kit to concentrate BACE-2. Avoid excessive boiling during sample preparation; heat samples at 70°C for 10 minutes rather than 95°C to prevent aggregation of hydrophobic membrane proteins. Load 30-50 µg per lane for whole-cell lysate or 10-20 µg for membrane fractions. Include reducing agent (DTT or β-mercaptoethanol) in sample buffer, as BACE-2 contains disulfide bonds that must be reduced for proper migration.

How should I store this BACE-2 antibody to maintain long-term stability?

Store at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce antibody titre and increase aggregation. The antibody is supplied in a glycerol-containing buffer that remains liquid at -20°C, allowing direct pipetting without thawing. For short-term use over 2-4 weeks, storing at 4°C is acceptable. Do not store diluted antibody; prepare working dilutions fresh for each experiment. Addition of 0.02% sodium azide as preservative is compatible with Western blot and IHC applications but will interfere with HRP-based detection if azide is carried through to the final incubation steps.