Western blot analysis of Beta Amyloid was performed using membrane enriched extract (30 ug lysate) of PC-3 (Lane 1) and tissue extract (30 ug lysate) of Rat Brain (Lane 2). The blots were probed with Anti-Beta Amyloid Mouse Monoclonal Antibody (Product # AHB0272, 2 µg/ml) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/ml, 1:2500 dilution). A 125 kDa isoform corresponding to Beta Amyloid was observed in PC-3 and, another isoform of 34 kDa was observed in Rat Brain. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
|Tested species reactivity||Human, Rat|
|Host / Isotype||Mouse / IgG1, kappa|
|Immunogen||The immunogen used in the production of this monoclonal antibody is a chemically synthesized peptide corresponding to amino acid residues 1-20 of A-beta (beta-amyloid) peptide.|
|Storage buffer||PBS, pH 7.2|
|Contains||0.1% sodium azide|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay Dependent|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Amyloid beta peptide is the major constituent of amyloid plaques in the brains of individuals afflicted with Alzheimer and quote;s disease. This peptide is generated from the beta-amyloid precursor protein (beta APP) in a two-step process. The first step involves cleavage of the extracellular, amino-terminal domain of beta APP. Protein cleavage is performed by an aspartyl protease termed beta-secretase (BACE). This enzyme is synthesized as a propeptide that must be modified to the mature and active form by the prohormone convertase, furin. Beta APP cleavage by the mature form of BACE results in the cellular secretion of a segment of beta APP and a membrane-bound remnant. This remnant is then processed by another protease termed gamma-secretase. Gamma-secretase cleaves an intra-membrane site in the carboxyl-terminal domain of beta APP, thus generating the amyloid beta peptide. Gamma-secretase is believed to be a multi-subunit complex containing presenilin-1 and 2 as central components. Found associated with the presenilins is the transmembrane glycoprotein nicastrin. Nicastrin has been found to bind to the carboxyl-terminus of betaAPP and helps to modulate the production of the amyloid beta peptide.
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