Human BID (BH3 Interacting Domain Death Agonist) ELISA Kit (HUES01753)

SKU:
HUES01753
€539

Description

ELISA Kit Technical ManualMSDS

Human BID (BH3 Interacting Domain Death Agonist) ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human BID . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human BID and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human BID, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm 2 nm. The OD value is proportional to the concentration of Human BID. The concentration of Human BID in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection MethodColormetric
Detection Range0.31-20 ng/mL
Sensitivity0.19 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human BID in samples. No significant cross-reactivity or interference between Human BID and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration
(ng/mL)
O.D Average Corrected
202.3
2.318
2.3092.225
101.6
1.636
1.6181.534
50.942
0.924
0.9330.849
2.50.417
0.445
0.4310.347
1.250.263
0.243
0.2530.169
0.630.191
0.173
0.1820.098
0.310.127
0.141
0.1340.05
00.082
0.086
0.084--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human BID were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human BID were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)1.031.878.591.091.979.01
Standard deviation0.060.080.390.060.090.37
C V (%)5.834.284.545.504.574.11

Recovery

The recovery of Human BID spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)95-106100
EDTA plasma (n=5)87-10095
Cell culture media (n=5)95-108101

Linearity

Samples were spiked with high concentrations of Human BID and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)96-110100-11297-111
Average (%)102106104
1:4Range (%)88-10284-9586-97
Average (%)949092
1:8Range (%)91-10682-9585-98
Average (%)978791
1:16Range (%)91-10683-9383-94
Average (%)978888

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human BID (BH3 Interacting Domain Death Agonist) ELISA Kit (HUES01753) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100 µL of standard solutions into the standard wells.
    3. Add 100 µL of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100 µL of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37 °C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100 µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37 °C.
    7. Aspirate or decant the solution from the plate and add 350 µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100 µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37 °C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90 µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37 °C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human BID (BH3 Interacting Domain Death Agonist) ELISA Kit (HUES01753) Protein Information

UniProt Protein Function:BID: a pro-apoptotic member of the Bcl-2 superfamily. Targets intracellular membranes and contains a BH3 death domain. Heterodimerizes with either the pro-apoptotic protein BAX or the anti-apoptotic protein BCL2, antagonizing its protective effect. The activity of BID is regulated by Caspase 8-mediated cleavage, exposing the BH3 domain and significantly changing the surface charge and hydrophobicity, causing translocation to mitochondria where it triggers cytochrome c release. Multiple alternatively spliced variants have been found.
UniProt Protein Details:

Protein type:Mitochondrial; Apoptosis

Chromosomal Location of Human Ortholog: 22q11.1

Cellular Component: cytosol; membrane; mitochondrial outer membrane; mitochondrion

Molecular Function:death receptor binding; protein binding

Biological Process: apoptotic mitochondrial changes; DNA damage response, signal transduction; induction of apoptosis via death domain receptors; neuron apoptosis; positive regulation of apoptosis; positive regulation of protein homooligomerization; positive regulation of protein oligomerization; regulation of apoptosis; release of cytochrome c from mitochondria

NCBI Summary:This gene encodes a death agonist that heterodimerizes with either agonist BAX or antagonist BCL2. The encoded protein is a member of the BCL-2 family of cell death regulators. It is a mediator of mitochondrial damage induced by caspase-8 (CASP8); CASP8 cleaves this encoded protein, and the COOH-terminal part translocates to mitochondria where it triggers cytochrome c release. Multiple alternatively spliced transcript variants have been found, but the full-length nature of some variants has not been defined. [provided by RefSeq, Jul 2008]
UniProt Code:P55957
NCBI GenInfo Identifier:2493285
NCBI Gene ID:637
NCBI Accession:P55957.1
UniProt Secondary Accession:P55957,Q549M7, Q71T04, Q7Z4M9, Q8IY86,
UniProt Related Accession:P55957
Molecular Weight:11,263 Da
NCBI Full Name:BH3-interacting domain death agonist
NCBI Synonym Full Names:BH3 interacting domain death agonist
NCBI Official Symbol:BID
NCBI Official Synonym Symbols:FP497
NCBI Protein Information:BH3-interacting domain death agonist
UniProt Protein Name:BH3-interacting domain death agonist
UniProt Synonym Protein Names:p22 BID; BID
Protein Family:BH3-interacting domain death agonist
UniProt Gene Name:BID
UniProt Entry Name:BID_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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