Unione® TR-FRET Human TSLP/TSLPR Binding Kit

Catalog Number: UA086125 Brand: UA BIOSCIENCE

Price:

$730 USD

Size: 100T 500T 2500T 10000T

Quantity:

For shipping services or bulk orders, you may request a quotation.

Secure checkout with

View full details

Product Details

Product Specification

Host	Human
Stability & Storage	-80℃

Background

[Background Introduction]

The kit employs homogeneous time-resolved fluorescence technology (TR-FRET) to measureTSLPand its interaction withTSLPR. This method enables simple and rapid high-throughput screening of inhibitors and antibody blockers.

As shown in the figure,the interaction betweenTSLPandTSLPRis detected using an Eu-labeled anti-Tag1 antibody (TR-FRET donor) and an Ac-labeled anti-Tag2 antibody (TR-FRET acceptor). BecauseTSLPandTSLPRbind, bringing the donor antibody close to the acceptor antibody, excitation of the donor antibody triggers fluorescence resonance energy transfer (FRET) to the acceptor antibody, causing the acceptor antibody to emit a signal at a wavelength of 665 nm.BosakitugA positive drug can blockTSLPandTSLPRfrom binding, preventing the FRET signal from being produced; thus, the screened drug targets TSLPandTSLPRThe stronger the blocking effect, the lower the signal.This specific signal is related toTSLPandTSLPRis proportional to the degree of their interaction. This homogeneous assay is easy to perform and requires no washing steps.

Components

[Product Componentsand Storage Conditions]

Components	Concentration	100T	500T	2500T	10000T	Storage Temperature
Tag1-TSLPRprotein	100×	5μL	20μL	100μL	400μL	-80℃
Tag2-TSLPprotein	100×	5μL	20μL	100μL	400μL	-80℃
Anti-Tag1 Eu antibody	100×	5μL	25μL	125μL	500μL	-80℃
Anti-Tag2 Ac antibody	25×	20μL	100μL	500μL	2000μL	-80℃
Detection buffer	10×	400μL	2mL	10mL	40mL	-80℃

Caution: After the first thaw, immediately aliquot and store at the recommended temperature. Avoid storing diluted samples or subjecting them to repeated freeze-thaw cycles.

Protocol

[Experimental Procedure and Operations]

1,Reagent Preparation

1.1Before use, thaw all reagents at room temperature (allow them to equilibrate at room temperature for at least 30 minutes). The reaction system in a 384-well plate is 20μL (the amount of reagents for the reaction system is shown in the table). Calculate the required volume before preparation and prepare accordingly; the following preparation is for reference only, using 500 reactions as an example.

Table 1.Reagent Preparation

Reagent Name	Configuration	Amount per Detection Well (μL)
Detection buffer	Take 2mL of 10× Detection Add 18 mL of deionized water to the buffer, diluteto1×, mixthoroughly and set asidefor use.-	Tag1-
TSLPRproteinTake 2	0μL of Tag1-TSLPRprotein stock solution,1×diluted with Detection bufferto2mL ,mix well and set asidefor use.	4
Tag2-TSLPprotein	take 20μL of Tag2-TSLPprotein stock solution, diluted 1×with Detection bufferto2mL ,Mix well and set aside.	4
Mix	Take25μL of Anti-Tag1 Eu antibody stock solution, add 1×Detection buffer 2.475mL,mix well and set aside; take100 μL of Anti-Tag2Acantibody stock solution, add 1×Detection buffer 2.4mL,mix well and set aside; Anti-Tag1 Eu antibody and Anti-Tag2Acantibody are each diluted and then mixed 1:1mix well and set aside.	10

1.2sample to be testedgradient dilution

takingBosakitugas an example, the diluent is 1× Detection buffer,to reduce the influence of matrix effect interference, it is recommended to use a solution with the same matrix as the sample to be tested for dilution; andthe sample to be tested should be adjusted according to its actual concentration.

Table2.Positive drugBosakitugGradient dilution(adjusted according to the actual situation)

	BosakitugFinal concentrationnM	BosakitugConfiguration concentrationnM	Preparation method
①	10.00	100.00	3μL 2μMBosakitug+57μL 1× Detection buffer
②	5.00	50.00	30μL ①+30μL 1× Detection buffer
③	2.50	25.00	30μL ②+30μL 1× Detection buffer
④	1.25	12.50	30μL ③+30μL 1× Detection buffer
⑤	0.63	6.25	30μL ④+30μL 1× Detection buffer
⑥	0.31	3.13	30μL ⑤+30μL 1× Detection buffer
⑦	0.16	1.56	30μL ⑥+30μL 1× Detection buffer
⑧	0.08	0.78	30μL ⑦+30μL 1× Detection buffer
⑨	0.04	0.39	30μL ⑧+30μL 1× Detection buffer
Blank	0	0	30μL 1× Detection buffer

2,Sample addition and controls

2.1 Sample addition order for experimental wells: 2 μL of the sample to be tested, 4 μLTag2-TSLPproteinworking solution, 4 μLTag1-TSLPRproteinworking solution, a mixed solution of 10 μL, added sequentially into the 384-well plate;

2.2maximum valuecontrol: 2 μL of 1× Detection buffer dilutedliquid,4 μLTag2-TSLPproteinworking solution, 4 μLTag1-TSLPRproteinworking solution, a mixed solution of 10 μL,added sequentially into the 384-well plate;

2.3 Quality control well (NC):10μL of 1× Detection buffer plus10μLMix.

After all samples have been added, centrifuge, cover with a plate seal, and incubate at room temperature1hour.

	Maximum valueControl	Sample	NC
Step one	2 μL 1× DetectionBuffer	2μL Gradient dilution of the sample to be tested	10uL 1× DetectionBuffer
	4 μLTag2-TSLPproteinworking solution
	incubate for 10 min
Step two	4 μLTag1-TSLPRproteinworking solution
	10μLMix
Step three	seal the plate with a membrane, incubate at room temperature1h

3,detection

Detect on an ELISA reader compatible with TR-FRET.Excitation light is 320/340 nm, detecting emission wavelengths at 620 nm and 665 nmemissionwavelengths.

[Result Calculation]

1)Calculate the signal value(Ratio): The fluorescence signal at 665 nm divided by the fluorescence signal at 620 nm, then multiplied by 10,000.

Ratio = (665/620)×10000

2)Calculated based on the signal valueNet signal:

Net signal= (Std-NC)/NC×100

3)Calculate CV (%):

CV (%) = Standard Deviation/Mean Ratio × 100%

[Data Example]

The following data cannot replace the data obtained in experiments; it is only for illustration, and the results maydepend on the plate readerinstrumentvaries.

Note: Recommendedmicroplate (384-well plate, white, shallow wells)

Country/region

Unione® TR-FRET Human TSLP/TSLPR Binding Kit