Description

Nori Human BAX ELISA Kit Summary

Alternative names for BAX: BCL2-associated X protein, Bcl-2-like protein 4

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Apoptosis regulator BAX, also known as BCL2-associated X protein (BAX) and bcl-2-like protein 4, is a protein that is encoded by the BAX gene.[1] BAX is a member of the Bcl-2 gene family. BAX forms a heterodimer with BCL2, and functions as an apoptotic activator. BAX is reported to interact with, and increase the opening of, the mitochondrial voltage-dependent anion channel (VDAC), ^[2] which leads to the loss in membrane potential and the release of cytochrome c. The expression of BAX gene is regulated by the tumor suppressor P53 and has been shown to be involved in P53-mediated apoptosis. In particular, p53 interacts with BAX, promoting its activation as well as its insertion into the mitochondrial membrane.^[3][4] In healthy mammalian cells, the majority of BAX is found in the cytosol, but upon initiation of apoptotic signaling, Bax undergoes a conformational shift. Upon induction of apoptosis, BAX becomes organelle membrane-associated, and in particular, mitochondrial membrane associated.^[5] Activated BAX and/or Bak may form an oligomeric pore, MAC in the MOM.^[6] This results in the release of cytochrome c and other pro-apoptotic factors from the mitochondria, often referred to as mitochondrial outer membrane permeabilization, leading to activation of caspases.^[7] This defines a direct role for BAX in mitochondrial outer membrane permeabilization. BAX activation is stimulated by various abiotic factors, including heat, hydrogen peroxide, low or high pH, and mitochondrial membrane remodeling. In addition, it can become activated by binding BCL-2, as well as non-BCL-2 proteins such as p53 and Bif-1. Conversely, BAX can become inactivated by interacting with VDAC2, Pin1, and IBRDC2.^[8]

References

1. Apte SS, et al. (1995) Genomics 26 (3), 592-594.
2. Shi Y, et al. (2003). Biochem. Biophys. Res. Commun. 305 (4): 989–96.
3. Miyashita T, et al. (1994). Oncogene. 9 (6): 1799–805. PMID8183579.
4. Miyashita T, Reed JC (1995). Cell. 80 (2): 293–9.
5. Hsu YT, et al. (1997). Proc. Natl. Acad. Sci. U.S.A. 94 (8): 3668–72.
6. McArthur, Kate; et al. (2018). Science. 359 (6378): eaao6047.
7. Weng C, et al. (2005). J. Biol. Chem. 280 (11): 10491–500.
8. Westphal, D; et al. (2014). Cell Death & Differentiation. 21 (2): 196–205.

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