Human Bruton'S Tyrosine Kinase ELISA Kit,Absin_specification/price/image

Product Detail
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Product Specification

Usage

Sample collection, processing and storage methods:
1, serum: use do not contain pyrogen and endotoxin in vitro, in the process of operation to avoid any cell stimulation, collect blood after 3000 turn centrifugal 10 minutes to separate serum and erythrocyte quickly carefully.
2, plasma: EDTA, citric acid salt or heparin anticoagulation. The supernatant was removed by centrifugation at 3000 RPM for 30 min.
3, on the cell supernatant: 3000 turn 10 minutes of centrifugal to get rid of particle and polymer.
4. Tissue homogenate: add appropriate amount of normal saline to the tissue and mash it. 3000 turn centrifugal take supernatant 10 minutes.
5. Storage: if the sample is not detected in time after collection, please pack according to the dosage, freeze at -20℃, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is evenly and fully thawed.
Bring your own items:
1. Microplate reader (450nm)
2 washer, high precision and spear: 0.5-10 ul, 2-20 ul, 20-200 - ul, ul 200-1000
3, 37℃ incubator
The reagent preparation:
Dilution of 20× wash buffer: Distilled water was diluted 1:20, that is, 1 part of 20× wash buffer was added to 19 parts of distilled water.
Washing plate method:
1, hand washing board: jilt liquid in the hole, every hole filled with liquid detergent. Let stand for 1 min after the left hole all liquid, pat dry on blotting paper, so wash board 5 times.
2. Automatic washing machine: inject 350μL of washing solution into each well, soak for 1min, and wash the plate 5 times.
Operation steps:
1. Remove the required slats from the aluminum foil bag after 20min of equilibrium at room temperature, and the remaining slats are sealed with a ziplack and placed back at 4 ° C.
Bore 2, set up the standard and sample, standard holes with different concentrations of standard 50 mu L;
3. Add 10μL sample to the sample well, and then add 40μL sample diluent; Blank holes are not added.
4, in addition to the blank hole, bore standard and sample of each hole to join horseradish peroxidase (HRP) labeled 100 mu L detection antibody, seal plate film sealed reaction with hole, 37 ℃ water bath pot or constant temperature box temperature 60 min.
5, abandon to liquid, pat dry on the blotting paper, every hole filled with liquid detergent. Let stand for 1 min, jilt to detergents, pat dry on the blotting paper, repeat 5 times wash plate (also available microplate wash plate).
6, 50μL of substrate A and B were added to each well and incubated at 37 ° C in the dark for 15min.
7, 50 mu, L per hole to join terminated liquid within 15 min, determine the hole in the 450 - nm wavelength, OD value.
Results:
Drawing standard curve: in Excel worksheets, with standard concentration as the abscissa, ordinate corresponding OD value, draw the standard linear regression curve, the curve equation of the sample density is calculated.

Theory

Double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used. Samples, standards, and HRP-labeled detection antibodies were added to the coated microwells precoated with Bruton's tyrosine kinase (Btk) antibody in turn, then incubated and washed thoroughly. After incubation and thorough washing, the substrate TMB was used for color development. TMB was converted to blue under the catalysis of peroxidase and to yellow under the action of acid. The degree of color is positively correlated with the level of Bruton's tyrosine kinase (Btk) in the sample. The activity of the samples was calculated by measuring the absorbance (OD) value at 450nm using a microplate reader.

Detection Type

Serum, cell supernatant, and tissue homogenate urine

Description

Bruton's tyrosine kinase (Btk or BTK for short), also known as tyrosine protein kinase BTK, is a tyrosine kinase encoded by the BTK gene. BTK plays a crucial role in B cell development. BTK contains five different protein interaction domains. These domains include an amino-terminal pleckstrin homology (PH) domain, a proline-rich TEC homology (TH) domain, the SRC homology (SH) domains SH2 and SH3, and a kinase structure with enzymatic activity. BTK plays a critical role in B cell development because it is required to transmit signals from pre-B cell receptors formed after successful immunoglobulin heavy chain rearrangement. Btk contains a PH domain that binds to phosphatidylinositol-triphosphate (PIP3). Binding of PIP3 induces Btk to phosphorylate phospholipase C, which in turn hydrolyses PIP2 (a phosphatidylinositol) to two second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG), which then regulate the activity of downstream proteins during B-cell signaling.

kit performance:
1. Accuracy: linear regression of standard and expected concentration correlation coefficient R value, greater than or equal to 0.9900.
2, Sensitivity: the minimum detection concentration is less than 10 U/mL.
3, specificity: does not cross-react with other soluble structural analogues.
4, repeatability: the coefficient of variation of intra-and inter-plate was less than 15%.

Composition

Name	96T	Note
Microwell microplate	12 holes×8
standard substance	0.3mL×6
Sample diluent	6mL
Detect antibody - HRP	10mL
20 x washing buffer	25mL	According to the instruction manual dilution
substrate A	6mL
substrate B	6mL
stop buffer	6mL
sealing film	2
ziplock bag	1

Note: The concentrations of standard substance (S0-S5) were 0, 75, 150, 300, 600, 1200 U/mL

General Notes

1. The kit was stored at 2-8 ° C and equilibrated at room temperature for 20 minutes before use. The concentrated washing solution removed from the refrigerator will have crystallization, which is a normal phenomenon, and the water bath will be heated to completely dissolve the crystals before use.
2. The slats that are not used in the experiment should be put back into the ziplock bag immediately, sealed (dry at low temperature) and stored.
3, concentration of 0 S0 standard can be regarded as negative control or blank; When operating according to the instructions, the sample has been diluted 5 times, and the final result multiplied by 5 is the actual concentration of the sample.
4, in strict accordance with the instructions indicated in the time, the amount of liquid and the order of the incubation operation.
5, all liquid components shake well before use.

Storage Temp.

2-8℃, protected from light and moisture, valid for 6 months.

Test Range

0.625-20 ng/mL

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.