Image 1: Bcl-2 target protein structure.

Bcl-2 Target Introduction

Protein Function

• Bcl-2 is an apoptosis inhibitor that can suppress apoptosis in various cellular systems.Bcl-2 can control mitochondrial membrane permeability to regulate cell death. It inhibits caspase activity by suppressing the release of mitochondrial cytochrome C or by binding to apoptotic activators.
• Bcl-2 also acts as an autophagy inhibitor, interacting with BECN1 and AMBRA1 to inhibit their autophagy functions.
• Bcl-2 weakens the activation of NLRP1 inflammasomes, reducing caspase 1 activation and IL1β release to alleviate inflammation.

Protein Expression

• Bcl-2 is expressed in various tissue cells.

Protein Characteristics

• Bcl-2 exists in two isoforms in humans and mice, with molecular weights of 26 kDa and 22 kDa, respectively.
• Bcl-2 can form homodimers or heterodimers.

Protein Localization

• Bcl-2 is mainly localized to the nuclear membrane, endoplasmic reticulum membrane, and mitochondrial outer membrane.

Figure 2: Bcl-2 ICC experimental result image, Anti-Bcl-2 antibody (ab194583)
Red: Bcl-2, Blue: DAPI.

Isoforms & post-translation modifications

• Human (P10415): Isoforms 1-2; 22-26 kDa (predicted)
• Mouse (P10417): Isoforms 1-2; 22-26 kDa (predicted)
• Rat (P49950): 27 kDa (predicted)
• Bcl-2 contains multiple phosphorylation sites, including Thr56, Ser70, Thr74, and Ser87.
• Phosphorylation/dephosphorylation of the Ser70 site regulates the anti-apoptotic activity of Bcl-2.
• Phosphorylation of the Thr69, Ser70, and Ser87 sites stimulates starvation-induced autophagy.
• Bcl-2 can be cleaved by caspases during the process of cell apoptosis, resulting in fragments that are 34 amino acids shorter than the full length and approximately 3 kDa smaller. The cleaved protein lacks the BH4 domain, leading to pro-apoptotic activity and the release of cytochrome C into the cytoplasm, further promoting caspase activity.
• Ubiquitination of Bcl-2 by PRKN increases protein stability; ubiquitination by SCF(FBXO10) leads to its degradation by the proteasome.

WB experiment tips

Precautions

• Bcl-2 exists in two isoforms in humans and mice, with molecular weights of 26 kDa and 22 kDa, respectively. Multiple bands may be detected in the WB experiment.
• Bcl-2 may be cleaved by caspases by approximately 3 kDa during apoptosis activation. In the WB experiment, the detected band size may not be completely consistent with the predicted protein size.
• Due to phosphorylation modification, the band size detected by WB may be different from the predicted value.
• Bcl-2 can form homodimers or heterodimers, so if not fully denatured, polymer forms may be detected, and the detected band size may not match the prediction.
• Bcl-2 is a relatively small protein, and it is recommended to use 12% to 15% concentration SDS-PAGE in WB experiments to obtain ideal results.

Positive control

• THP1 cell lysate.

Example of results

Figure 3: Recombinant Anti-Bcl-2 antibody [EPR17509] (ab182858)

Lane 1: Wild-type HAP1 whole cell lysate.
Lane 2: BCL2 knockout HAP1 whole cell lysate.
Lane 3: HeLa whole cell lysate.
Lane 4: THP-1 whole cell lysate.

Result description: Bcl-2 (green), GAPDH (red).
Predicted band size: 26 kDa

Key control points

In the experiment, attention should be given to key control points in addition to routine issues:

Sample preparation:

1. We recommend using freshly prepared samples, do not freeze, to prevent protein degradation.
2. Add a cocktail of protease inhibitors to prevent degradation of the target protein.
3. Keep the sample on ice throughout the sample preparation process.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.

Transferring:

1. We recommend using Coomassie Brilliant Blue staining after transferring to confirm the success of the transfer.
2. For target proteins with smaller molecular weights, it is recommended to use a 0.22 μm PVDF membrane and avoid over-transferring.
3. We recommend keeping the entire membrane intact for antibody incubation.

Antibody incubation:

1. Please choose the appropriate antibody working concentration according to the product manual.

References

1. Waseem Ahmad Siddiqui 1, Amjid Ahad, Haseeb Ahsan. The mystery of BCL2 family: Bcl-2 proteins and apoptosis: an update. Arch Toxicol. 2015 Mar;89(3):289-317. doi: 10.1007/s00204-014-1448-7. Epub 2015 Jan 25.
2. D T Chao, S J Korsmeyer. BCL-2 family: regulators of cell death. Annu Rev Immunol. 1998;16:395-419. doi: 10.1146/annurev.immunol.16.1.395.
3. P P Ruvolo 1, X Deng, W S May. Phosphorylation of Bcl2 and regulation of apoptosis. Leukemia. 2001 Apr;15(4):515-22. doi: 10.1038/sj.leu.2402090.
4. Yiping Rong, Clark W Distelhorst. Bcl-2 protein family members: versatile regulators of calcium signaling in cell survival and apoptosis. Annu Rev Physiol. 2008;70:73-91. doi: 10.1146/annurev.physiol.70.021507.105852.
5. Avi Ashkenazi, Wayne J Fairbrother, Joel D Leverson, Andrew J Souers. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov. 2017 Apr;16(4):273- 284.doi: 10.1038/nrd.2016.253.