Figure 2: The relationship between protein concentration of lysates from HeLa cells, untransfected or transfected with a construct expressing BACE protein, and the absorbance at 450 nm as detected by the PathScan® Total BACE Sandwich ELISA Kit is shown.
Figure 1: BACE protein was detected from rat brain lysate and HeLa cell lysates, untransfected (-) or transfected with a construct expressing BACE protein (+), using the PathScan® Total BACE Sandwich ELISA Kit #13907. The absorbance readings at 450 nm are shown in the top figure, while the corresponding western blot using BACE (D10E5) Rabbit mAb #5606 is shown in the lower figure.
|Product Includes||Volume||Solution Color|
|BACE Rabbit mAb Coated Microwells||96 tests|
|BACE1 Mouse Detection mAb||1 ea||Green (Lyophilized)|
|Anti-mouse IgG, HRP-linked Antibody (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
PathScan® Total BACE Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous and transfected levels of BACE protein. A BACE Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, BACE protein is captured by the coated antibody. Following extensive washing, a BACE Mouse Detection mAb is added to detect the captured BACE protein. Anti-mouse IgG, HRP-linked Antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of BACE protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
PathScan® Total BACE Sandwich ELISA Kit detects endogenous and transfected levels of BACE protein as shown in Figure 1. The kit sensitivity is shown in Figure 2. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
β-secretase (BACE) is an aspartic acid proteinase that catalyses the initial step in APP processing by cleaving and releasing a soluble, extracellular APP-β (sAPPβ) ectodomain and generating a membrane-bound, carboxy-terminal fragment consisting of 99 amino acids (CTF99). Additional processing of CTF99 by γ-secretase generates the amyloid β-peptide (Aβ) that forms aggregates in the brains of Alzheimer disease (AD) patients. BACE is an attractive target for inhibitors in AD therapy since it catalyses the first and rate limiting step in amyloidogenic APP processing (1). Pro-BACE-1 is synthesized in the ER before it is transported to the trans-Golgi network to undergo maturation (2). BACE-1 functions as a dimer and is phosphorylated at Ser498 by casein kinase 1, which is necessary for recycling back to the membrane from the early endosome (3,4).
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