Mouse Bcl2 antagonist of cell death (Bad) ELISA Kit
The Mouse Bcl2-Antagonist of Cell Death (BAD) ELISA Kit is specifically designed for the quantitative detection of BAD levels in mouse serum, plasma, and cell lysates. With its high sensitivity and specificity, this kit ensures accurate and reliable results, making it a valuable tool for researchers studying cell apoptosis and survival pathways.BAD is a key regulator of apoptosis, playing a critical role in cell death and survival processes. Dysregulation of BAD has been implicated in various diseases, including cancer, neurodegenerative disorders, and autoimmune conditions.
By accurately measuring BAD levels, researchers can gain valuable insights into the mechanisms underlying these diseases and potentially identify new therapeutic targets.Overall, the Mouse Bcl2-Antagonist of Cell Death (BAD) ELISA Kit provides researchers with a powerful tool for studying the role of BAD in health and disease, offering a comprehensive and reliable method for quantifying BAD levels in mouse samples.
Product Name:
Mouse Bcl2 antagonist of cell death (Bad) ELISA Kit
SKU:
MOEB1440
Size:
96T
Target:
Mouse Bcl2 antagonist of cell death (Bad)
Synonyms:
Bcl-2-binding component 6, Bcl-xL/Bcl-2-associated death promoter, Bcl2 antagonist of cell death, BAD, Bbc6
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.312-20ng/mL
Sensitivity:
0.12ng/mL
Intra CV:
0.0%
Inter CV:
0.0%
Linearity:
Sample
1:2
1:4
1:8
1:16
Serum(N=5)
109-118%
99-107%
87-87%
110-120%
EDTA Plasma(N=5)
99-109%
106-115%
112-121%
95-107%
Heparin Plasma(N=5)
108-117%
81-90%
105-113%
88-100%
Recovery:
Sample Type
Average(%)
Recovery Range(%)
Serum
80
80-80
Plasma
80
80-80
Function:
Promotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. Can reverse the death repressor activity of Bcl-X(L), but not that of Bcl-2. Appears to act as a link between growth factor receptor signaling and the apoptotic pathways.
Uniprot:
Q61337
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Bcl2-associated agonist of cell death
Sub Unit:
Forms heterodimers with the anti-apoptotic proteins, Bcl-X(L), Bcl-2 and Bcl-W. Also binds protein S100A10 (By similarity). The Ser-112/Ser-136 phosphorylated form binds 14-3-3 proteins. Interacts with AKT1 and PIM3 (By similarity). Interacts (via BH3 domain) with NOL3 (via CARD domain); preventing the association of BAD with BCL2 (By similarity). Interacts with HIF3A isoform 2 (via C-terminus domain); the interaction reduces the binding between BAD and BAX (PubMed:21546903).
Research Area:
Cancer
Subcellular Location:
Mitochondrion outer membrane Cytoplasm Colocalizes with HIF3A isoform 2 in the cytoplasm (PubMed:21546903). Upon phosphorylation, locates to the cytoplasm.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
BAD: a proapoptotic member of the Bcl-2 family. Displaces Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death. Survival factors such as IL-3 can inhibit the apoptotic activity of Bad inducing the phosphorylation of Bad by Akt and p90RSK. 14-3-3 proteins bind phosphorylated Bad, inhibiting its binding to Bcl-2 and Bcl-xL. Phosphorylation by mitochondria-anchored PKA in the BH3 domain can block the dimerization of Bad and Bcl-xL.
Molecular Function:protein kinase B binding; protein binding; protein heterodimerization activity; phospholipid binding; caspase activator activity; protein kinase binding; lipid binding; protein phosphatase binding; protein phosphatase 2B binding
Biological Process: positive regulation of proteolysis; apoptosis; positive regulation of apoptosis; positive regulation of caspase activity; regulation of caspase activity; cellular process regulating host cell cycle in response to virus; glucose homeostasis; positive regulation of apoptosis by virus; regulation of apoptosis; pore complex biogenesis; positive regulation of glucokinase activity; caspase activation; release of cytochrome c from mitochondria; cytokine and chemokine mediated signaling pathway; suppression by virus of host apoptosis; ADP metabolic process; positive regulation of insulin secretion; ATP metabolic process; glucose catabolic process; cell proliferation; positive regulation of T cell differentiation; induction of apoptosis via death domain receptors; DNA damage response, signal transduction resulting in induction of apoptosis; positive regulation of B cell differentiation; regulation of mitochondrial membrane permeability; positive regulation of epithelial cell proliferation
bcl2-associated agonist of cell death; bcl-2-binding component 6; bcl2 antagonist of cell death; bcl-xL/Bcl-2-associated death promoter
UniProt Protein Name:
Bcl2-associated agonist of cell death
UniProt Synonym Protein Names:
Bcl-2-binding component 6; Bcl-xL/Bcl-2-associated death promoter; Bcl2 antagonist of cell death
Protein Family:
Bcl2-associated agonist of cell death
UniProt Gene Name:
Bad
UniProt Entry Name:
BAD_MOUSE
Component
Quantity (96 Assays)
Storage
ELISA Microplate (Dismountable)
8×12 strips
-20°C
Lyophilized Standard
2
-20°C
Sample Diluent
20ml
-20°C
Assay Diluent A
10mL
-20°C
Assay Diluent B
10mL
-20°C
Detection Reagent A
120µL
-20°C
Detection Reagent B
120µL
-20°C
Wash Buffer
30mL
4°C
Substrate
10mL
4°C
Stop Solution
10mL
4°C
Plate Sealer
5
-
Other materials and equipment required:
Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
Step
1.
Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.
Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.
Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.
Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.
Repeat the wash process for five times as conducted in step 3.
6.
Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.
Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.
Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.
After experiment, store all reagents according to the specified storage temperature respectively until their expiry.
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
Sample Type
Protocol
Serum
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
