|Reactivity||Human Mouse Rat|
|Storage buffer||10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.|
|Storage||Store at –20°C.|
1. Aspirate media from cultures and Wash the cells with 1X PBS. 2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice. 3. Sonicate for 10–15 sec to complete cell lysis and shear DNA. 4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice. 5. Centrifuge for 5 min (with Microcentrifuge). 6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins). NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer. 7. Electrotransfer to nitrocellulose/PVDF membrane.
Membrane Blocking and Antibody Incubations
1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature. 2. Incubate the membrane in the blocking buffer for 1 hr at room temperature. 3. Wash three times for 5 min each with TBST.
b. Antibodies Incubation
1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C. 2. Wash three times for 5 min each with TBST. 3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature. 4. Wash three times for 5 min each with TBST. 5. Proceed with detection.
Detection of Proteins
1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST. 2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well. 3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.
|Specificity||BACE1 Rabbit Recombinant mAb detects endogenous levels of total BACE1.|
|Background||BACE1 is a type 1 transmembrane aspartic protease related to the pepsins and retroviral aspartic proteases. BACE1 activity has a low optimum pH, and the enzyme is predominantly localized in acidic intracellular compartments (for example, endosomes, trans-Golgi) with its active site in the lumen of the vesicle. The highest expression levels of BACE1 are found in neurons. BACE1 is phosphorylated on Ser 498, and this phosphorylation together with a C-terminal acidic cluster dileucine motif (DXXLL) regulates BACE1 recycling between the cell surface and endosomal compartments. In addition to APP, BACE1 has other substrates. BACE1 substrates are transmembrane proteins, such as Golgi-localized membrane-bound α2,6-sialyltransferase, IL-1 type II receptor, P-selectin glycoprotein ligand-1, APP homologs Aβ precursor-like protein-1 and Aβ precursor-like protein-2, low-density lipoprotein receptor-related protein, the voltage-gated sodium channel β1 to β4 subunits, neuregulin-1 and neuregulin-3.|