Anti-BACE1 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for BACE1 on Western blot?

Full-length mature BACE1 runs at approximately 70 kDa on reducing SDS-PAGE, reflecting the ~501 amino acid mature enzyme plus N-glycosylation at four sites. The pro-form (proBACE1) runs slightly higher at approximately 75 kDa before propeptide cleavage by furin. In brain and neuronal cell lysates, the mature 70 kDa band is dominant. Some cell types show additional bands corresponding to alternatively glycosylated or C-terminally processed forms in the 55-65 kDa range.

What is the recommended starting dilution for BACE1 Western blot?

Start at 1:1000 dilution in blocking buffer for whole cell lysates, mouse or human brain homogenates, and neuronal culture lysates that express BACE1 at biologically relevant levels. Load 20-30 µg total protein per lane. For low-expressing samples (peripheral tissues, transfected control cells at low copy number), you may need to concentrate to 1:500. For overexpression systems where BACE1 is at high copy, dilute to 1:2000 or 1:5000. Optimize by serial dilution across 4-5 points on your positive control lysate.

Does this antibody cross-react with BACE2 or other aspartic proteinases?

BACE1 and BACE2 share approximately 64% sequence identity in the protease domain. The domain-specific epitopes used across the TPB BACE1 Superpooled kit are chosen from regions where BACE1 diverges from BACE2 — specifically the propeptide region, the C-terminal cytoplasmic tail, and portions of the catalytic domain that lie outside the conserved catalytic aspartates. Cross-reactivity with BACE2 is minimal under standard Western blot conditions. Other aspartic proteinases (cathepsin D, cathepsin E, pepsin, renin) share only ~30% identity and are not detected.

What sample types and lysis buffers work best for detecting endogenous BACE1?

BACE1 is expressed most abundantly in neurons of the central nervous system. Adult brain cortex and hippocampus give strong signal. Cerebellar granule cells, dorsal root ganglia, and cultured primary cortical neurons work well. Peripheral tissues (pancreatic β-cells, kidney) show detectable but lower expression. RIPA buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% NP-40, 0.5% deoxycholate, 0.1% SDS) with protease inhibitor cocktail extracts BACE1 efficiently. Include an aspartic-protease inhibitor cocktail (pepstatin A at 1 µg/mL) if you're concerned about autoproteolysis during processing.

Is this antibody validated in mouse or rat brain tissue?

Mouse and rat BACE1 share approximately 92% and 90% sequence identity respectively with human. Cross-reactivity is expected based on sequence homology across the catalytic and cytoplasmic tail domains. Positive detection in adult mouse cortical lysate has been confirmed as a routine positive control. Rat detection is predicted but should be validated in your specific rat sample type before use as a primary reagent.

What positive and negative controls should I use for BACE1?

Positive controls: adult mouse brain cortical lysate (endogenous BACE1 expressed at appreciable levels), human SH-SY5Y neuroblastoma cells (endogenous), or recombinant BACE1 protein (available as REC-BACE1 from Triple Point Biologics). Negative controls: BACE1 knockout mouse cortex tissue (available from Jackson Laboratory, Bace1-/- mice are viable), or transient BACE1 siRNA knockdown in HEK293 cells (achieves ~80% reduction within 72 hours). Non-neural tissues that do not express BACE1 (skeletal muscle, skin epidermis) serve as additional negative controls.

Why do I see multiple bands between 55 and 75 kDa?

BACE1 exists in several biologically relevant molecular weight forms: the ~75 kDa proenzyme (with intact propeptide), the ~70 kDa mature form (post-furin cleavage), and shed soluble ectodomain forms (sBACE1) at ~55-60 kDa released by ectodomain shedding. Detection of multiple bands is expected and biologically informative. Using the Superpooled BACE1 kit (RP1BACE1 propeptide + RP2BACE1 catalytic + RP3BACE1 cytoplasmic tail) lets you distinguish these forms by which antibodies detect which bands — a domain-by-domain map of BACE1 processing state.

How should I store the antibody after opening?

Store the unopened vial at -20°C. After opening, aliquot into working volumes (10-50 µL depending on your experiment frequency) and store aliquots at -20°C in single-use format. Working aliquots can be kept at 4°C for up to one month with negligible potency loss. Avoid repeated freeze-thaw cycles — polyclonal potency decreases 10-15% per freeze-thaw. Do not store diluted working solutions at 4°C beyond one week.