Description
Bak Colorimetric Cell-Based ELISA Kit
The BAK Colorimetric Cell-Based ELISA Kit is a cutting-edge product designed for the precise measurement of BAK levels in cell cultures. This innovative kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.BAK is a key protein involved in regulating apoptosis, the process of programmed cell death. Dysregulation of BAK has been linked to various diseases such as cancer, autoimmune disorders, and neurological conditions, making it a valuable biomarker for studying these diseases and developing targeted therapies.
With the BAK Colorimetric Cell-Based ELISA Kit, researchers can confidently measure BAK levels in cell cultures with ease and precision, aiding in the advancement of scientific knowledge and the discovery of novel treatment strategies.
| Product Name: | Bak Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00052 |
| ELISA Type: | Cell-Based |
| Target: | Bak |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Bak Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Bak protein expression profile in cells. The kit can be used for measuring the relative amounts of Bak in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Bak.
Qualitative determination of Bak concentration is achieved by an indirect ELISA format. In essence, Bak is captured by Bak-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 578, UniProt ID: Q16611/Q13014, OMIM: 600516, Unigene: Hs.485139 |
| Gene Symbol: | BAK1 |
| Sub Type: | None |
| UniProt Protein Function: | BAK1: In the presence of an appropriate stimulus, accelerates programmed cell death by binding to, and antagonizing the anti- apoptotic action of BCL2 or its adenovirus homolog E1B 19k protein. Low micromolar levels of zinc ions inhibit the promotion of apoptosis. Interacts with BCL2A1. Homodimer. Formation of the homodimer is zinc-dependent. Forms heterodimers with BCL2, E1B 19k protein, and BCL2L1 isoform Bcl-X(L). Interacts with myxoma virus protein M11L. Expressed in a wide variety of tissues, with highest levels in the heart and skeletal muscle. Belongs to the Bcl-2 family. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Mitochondrial; Endoplasmic reticulum; Apoptosis Chromosomal Location of Human Ortholog: 6p21.3 Cellular Component: cytosol; endoplasmic reticulum; integral to mitochondrial outer membrane; mitochondrial outer membrane; mitochondrion; pore complex Molecular Function:BH domain binding; chaperone binding; heat shock protein binding; identical protein binding; metal ion binding; protein binding; protein heterodimerization activity; protein homodimerization activity Biological Process: aging; apoptosis; B cell apoptosis; B cell homeostasis; B cell negative selection; blood vessel remodeling; brain development; caspase activation via cytochrome c; cell proliferation; cytolysis; DNA damage response, signal transduction resulting in induction of apoptosis; endocrine pancreas development; endoplasmic reticulum calcium ion homeostasis; establishment and/or maintenance of transmembrane electrochemical gradient; limb morphogenesis; mitochondrial fusion; myeloid cell homeostasis; negative regulation of cell proliferation; negative regulation of peptidyl-serine phosphorylation; organ regeneration; positive regulation of apoptosis; positive regulation of proteolysis; post-embryonic camera-type eye morphogenesis; programmed cell death; reduction of endoplasmic reticulum calcium ion concentration; regulation of cell cycle; regulation of mitochondrial membrane permeability; regulation of mitochondrial membrane potential; regulation of protein heterodimerization activity; regulation of protein homodimerization activity; release of cytochrome c from mitochondria; response to drug; response to ethanol; response to fungus; response to gamma radiation; response to hydrogen peroxide; response to mycotoxin; response to organic cyclic substance; response to UV-C; unfolded protein response, activation of signaling protein activity; vagina development |
| NCBI Summary: | The protein encoded by this gene belongs to the BCL2 protein family. BCL2 family members form oligomers or heterodimers and act as anti- or pro-apoptotic regulators that are involved in a wide variety of cellular activities. This protein localizes to mitochondria, and functions to induce apoptosis. It interacts with and accelerates the opening of the mitochondrial voltage-dependent anion channel, which leads to a loss in membrane potential and the release of cytochrome c. This protein also interacts with the tumor suppressor P53 after exposure to cell stress. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q16611 |
| NCBI GenInfo Identifier: | 2493274 |
| NCBI Gene ID: | 578 |
| NCBI Accession: | Q16611.1 |
| UniProt Secondary Accession: | Q16611,Q6I9T6, Q92533, C0H5Y7, |
| UniProt Related Accession: | Q16611 |
| Molecular Weight: | 16,872 Da |
| NCBI Full Name: | Bcl-2 homologous antagonist/killer |
| NCBI Synonym Full Names: | BCL2 antagonist/killer 1 |
| NCBI Official Symbol: | BAK1 |
| NCBI Official Synonym Symbols: | BAK; CDN1; BCL2L7; BAK-LIKE |
| NCBI Protein Information: | bcl-2 homologous antagonist/killer |
| UniProt Protein Name: | Bcl-2 homologous antagonist/killer |
| UniProt Synonym Protein Names: | Apoptosis regulator BAK; Bcl-2-like protein 7; Bcl2-L-7 |
| UniProt Gene Name: | BAK1 |
| UniProt Entry Name: | BAK_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-Bak Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
