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Human BRCA1 ELISA Kit

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SKU:
HUFI00836
$681

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human BRCA1 ELISA Kit - Information

The ELISA Genie BRCA1 ELISA Kit can assay for BRCA1 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our BRCA1 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound BRCA1 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human BRCA1 ELISA Kit - Data

Description

E3 ubiquitin-protein ligase that specifically mediates the formation of 'Lys-6'-linked polyubiquitin chains and plays a central role in DNA repair by facilitating cellular responses to DNA damage. It is unclear whether it also mediates the formation of other types of polyubiquitin chains. The E3 ubiquitin-protein ligase activity is required for its tumor suppressor function. The BRCA1-BARD1 heterodimer coordinates a diverse range of cellular pathways such as DNA damage repair, ubiquitination and transcriptional regulation to maintain genomic stability. Regulates centrosomal microtubule nucleation. Required for normal cell cycle progression from G2 to mitosis. Required for appropriate cell cycle arrests after ionizing irradiation in both the S-phase and the G2 phase of the cell cycle. Involved in transcriptional regulation of P21 in response to DNA damage. Required for FANCD2 targeting to sites of DNA damage. May function as a transcriptional regulator. Inhibits lipid synthesis by binding to inactive phosphorylated ACACA and preventing its dephosphorylation. Contributes to homologous recombination repair (HRR) via its direct interaction with PALB2, fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Component of the BRCA1-RBBP8 complex which regulates CHEK1 activation and controls cell cycle G2/M checkpoints on DNA damage via BRCA1-mediated ubiquitination of RBBP8. Acts as a transcriptional activator (PubMed:20160719).1

Post-Translational Modification

Phosphorylation at Ser-308 by AURKA is required for normal cell cycle progression from G2 to mitosis. Phosphorylated in response to IR, UV, and various stimuli that cause checkpoint activation, probably by ATM or ATR. Phosphorylation at Ser-988 by CHEK2 regulates mitotic spindle assembly. Autoubiquitinated, undergoes 'Lys-6'-linked polyubiquitination. 'Lys-6'-linked polyubiquitination does not promote degradation.

Uniprot ID P38398
Alias

BRCA1/RNF53/Breast cancer type 1 susceptibility protein/RING finger protein 53

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of BRCA1 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.312-20ng/ml

Sensitivity

< 0.188ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of BRCA1 and the recovery rates were calculated by comparing the measured value to the expected amount of BRCA1 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 90-98 94
EDTA plasma(n=5) 93-104 99
UFH plasma(n=5) 90-102 95
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of BRCA1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 94-104% 89-101% 86-104% 85-97%
EDTA plasma(n=5) 82-95% 84-93% 84-101% 89-101%
UFH plasma(n=5) 82-97% 87-95% 80-93% 84-96%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Protocol

Sandwich ELISA Protocol

The below protocol is a sample protocol for a sandwich ELISA using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of analyte present in their sample. This can be very useful when looking for increases in protein concentration, phosphorylation of proteins or decreases on protein concentrations.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at room temperature (37 °C). When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

ELISA Kit Technical Manual

Procedure:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 15-30 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.
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Additional Information

Reactivity:
Human
ELISA Type:
Sandwich
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