Human BAG3 (Bcl2 Associated Athanogene 3) ELISA Kit (HUES01631)

SKU:
HUES01631
€539

Description

ELISA Kit Technical ManualMSDS

Human BAG3 (Bcl2 Associated Athanogene 3) 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 BAG3 . 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 BAG3 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 BAG3, 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 BAG3. The concentration of Human BAG3 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 Range0.16-10 ng/mL
Sensitivity0.10 ng/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids

Specificity

This kit recognizes Human BAG3 in samples. No significant cross-reactivity or interference between Human BAG3 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
102.462
2.502
2.4822.392
51.592
1.644
1.6181.528
2.50.903
0.901
0.9020.812
1.250.482
0.516
0.4990.409
0.630.301
0.289
0.2950.205
0.320.194
0.186
0.190.1
0.160.132
0.15
0.1410.051
00.085
0.095
0.09--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human BAG3 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human BAG3 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.491.194.730.471.105.17
Standard deviation0.030.070.210.030.070.18
C V (%)6.125.884.446.386.363.48

Recovery

The recovery of Human BAG3 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)85-9590
EDTA plasma (n=5)88-10496
Cell culture media (n=5)90-10497

Linearity

Samples were spiked with high concentrations of Human BAG3 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 (%)92-10591-10294-107
Average (%)9996102
1:4Range (%)91-10681-9387-99
Average (%)978792
1:8Range (%)85-10181-9287-99
Average (%)928793
1:16Range (%)93-10682-9280-95
Average (%)1008787

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 BAG3 (Bcl2 Associated Athanogene 3) ELISA Kit (HUES01631) 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 BAG3 (Bcl2 Associated Athanogene 3) ELISA Kit (HUES01631) Protein Information

UniProt Protein Function:BAG3: Inhibits the chaperone activity of HSP70/HSC70 by promoting substrate release. Has anti-apoptotic activity. Defects in BAG3 are the cause of myopathy myofibrillar type 6 (MFM6). A neuromuscular disorder that results in early-onset, severe, progressive, diffuse muscle weakness associated with cardiomyopathy, severe respiratory insufficiency during adolescence, and a rigid spine in some patients. At ultrastructural level, muscle fibers display structural alterations consisting of replacement of the normal myofibrillar markings by small, dense granules, or larger hyaline masses, or amorphous material. Defects in BAG3 are the cause of cardiomyopathy dilated type 1HH (CMD1HH). CMD1HH is a disorder characterized by ventricular dilation and impaired systolic function, resulting in congestive heart failure and arrhythmia. Patients are at risk of premature death.
UniProt Protein Details:

Protein type:Apoptosis

Chromosomal Location of Human Ortholog: 10q26.11

Cellular Component: cell-cell adherens junction; cytoplasm; cytosol; plasma membrane

Molecular Function:adenyl-nucleotide exchange factor activity; protein binding

Biological Process: induction of apoptosis via death domain receptors

Disease: Cardiomyopathy, Dilated, 1hh; Myopathy, Myofibrillar, 6

NCBI Summary:BAG proteins compete with Hip for binding to the Hsc70/Hsp70 ATPase domain and promote substrate release. All the BAG proteins have an approximately 45-amino acid BAG domain near the C terminus but differ markedly in their N-terminal regions. The protein encoded by this gene contains a WW domain in the N-terminal region and a BAG domain in the C-terminal region. The BAG domains of BAG1, BAG2, and BAG3 interact specifically with the Hsc70 ATPase domain in vitro and in mammalian cells. All 3 proteins bind with high affinity to the ATPase domain of Hsc70 and inhibit its chaperone activity in a Hip-repressible manner. [provided by RefSeq, Jul 2008]
UniProt Code:O95817
NCBI GenInfo Identifier:12643665
NCBI Gene ID:9531
NCBI Accession:O95817.3
UniProt Secondary Accession:O95817,Q3B763, Q9NT20, Q9P120, A8K5L8,
UniProt Related Accession:O95817
Molecular Weight:61,595 Da
NCBI Full Name:BAG family molecular chaperone regulator 3
NCBI Synonym Full Names:BCL2 associated athanogene 3
NCBI Official Symbol:BAG3
NCBI Official Synonym Symbols:BIS; MFM6; BAG-3; CAIR-1
NCBI Protein Information:BAG family molecular chaperone regulator 3
UniProt Protein Name:BAG family molecular chaperone regulator 3
UniProt Synonym Protein Names:Bcl-2-associated athanogene 3; Bcl-2-binding protein Bis; Docking protein CAIR-1
Protein Family:BAG family molecular chaperone regulator
UniProt Gene Name:BAG3
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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