Unione® TR-FRET Human IL17A/IL17RA Binding Kit

Catalog Number: UA086014 Brand: UA BIOSCIENCE

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$2,180 USD

Size: 500T 100T 2500T 10000T

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Product Details

Product Specification

Host	Human
Stability & Storage	-80℃

Background

The kit employs homogeneous time-resolved fluorescence technology.TR-FRET), used for the determination ofIL17AwithIL17RAthe interactions between them. This method enables simple and rapid high-throughput identification of inhibitors and antibody blockers.

As shown in the figure,IL17AwithIL17 RAThe interaction between them is achieved by usingIMarked anti-Tag1Antibody (TR-FRETDonor) andAcMarkedTag2Antibody (TR-FRET(receptor) testing. Due toIL17AwithIL17 RABy bringing the donor antibody and the acceptor antibody into close proximity, excitation of the donor antibody triggers fluorescence resonance energy transfer to the acceptor antibody.Förster resonance energy transfer), enabling the receptor antibody to emit specifically.665 nmA signal of a specific wavelength.SecukinumabPositive drugs can block.IL17AwithIL17 RAThe combination of, makesFRETThe signal cannot be generated, and the screened drug affects…IL17AwithIL17 RAThe stronger the blocking effect, the lower the signal. This specific signal is directly proportional to the extent of interaction betweenIL17AandIL17 RA.This homogeneous assay is easy to perform and requires no washing steps.

Components

Component	Concentration	100T	500T	2500T	10000T	Storage Temperature
Tag1- IL17A protein	100×	5μL	20μL	100μL	400μL	-80℃
Tag2- IL17 RA protein	100×	5μL	20μL	100μL	400μL	-80℃
Anti-Tag1 Eu antibody	50×	10μL	50μL	250μL	1000μL	-80℃
Anti-Tag2 Ac antibody	12.5×	40μL	200μL	1000μL	4000μL	-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

1,Reagent preparation

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

Table 1.Reagent preparation

Reagent name	Configuration	μL per test well
Detection buffer	Take 210 mLtimes Detection Add 18 mL of deionized water to the buffer, dilute.Explanationto1×, mixedEvenly prepareduse.	-
Sample to be tested	According to the reaction system, the sample to be tested is diluted with 1× Detection buffer to the desired concentration..If DMSO is used, ensure that the DMSO concentration is consistent across all assay wells.	2
Tag1- IL17A protein	take20 μL Tag1-IL17A proteinUndiluted solution,1× Detection BufferDilute to2 mL,Mix well and set aside.	4
Tag2- IL17 RA protein	take20 μL Tag2-IL17 RA proteinUndiluted solution,1× Detection BufferDilute to2 mL,Mix well and set aside.	4
Antibody Mix	take50 μL Anti-Tag1 Eu antibodyConcentrate, add1× Detection buffer 2.45 mL,Mix well and set aside.; take200 μL Anti-Tag2 Ac antibodyConcentrate, add1× Detection buffer 2.3 mL,Mix well and set aside.; Anti-Tag1 Eu antibodyandAnti-Tag2 Ac antibodyAfter separate dilutions1:1Mix well and set aside.	10

1.2Sample to be testedGradient dilution

withSecukinumabFor example, the diluent is 1× Detection buffer.To minimize the impact of matrix effects, it is recommended to dilute the sample with a solution that has the same matrix as the analyte; andThe sample to be tested should be adjusted according to its actual concentration.

Table2.Positive drug SsecukinumabGradient dilution(Adjust according to actual conditions)

	SecukinumabFinal concentrationnM	SecukinumabPreparation concentrationnM	Preparation method
①	100	1000	1μL100μMSsecukinumab+99μL 1× Detection buffer
②	33.33	333.33	20μL ①+40μL 1× Detection buffer
③	11.11	111.11	20μL ②+40μL 1× Detection buffer
④	3.70	37.04	20μL ③+40μL 1× Detection buffer
⑤	1.23	12.35	20μL ④+40μL 1× Detection buffer
⑥	0.41	4.12	20μL ⑤+40μL 1× Detection buffer
⑦	0.14	1.37	20muL ⑥+40muL 1× Detection buffer
⑧	0.05	0.46	20muL ⑦+40muL 1× Detection buffer
⑨	0.015	0.15	20muL ⑧+40muL 1× Detection buffer
Blank	0	0	40muL 1× Detection buffer

2,Sample addition and controls

2.1Sample addition order for the experimental wells:2 μLSample to be tested,4 μL Tag1-IL17A proteinWorking fluid,4 μL Tag2-IL17 RA proteinWorking fluid,10 μL Antibody Mix, add in sequence384In the shallow-hole plate;

2.2Maximum value reference hole:2 μL 1× Detection buffer,4 μL Tag1-IL17A proteinWorking fluid,4 μL Tag2-IL17 RA proteinWorking fluid,10 μL Antibody Mix, add in sequence384In the shallow-hole plate;

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

After all samples have been added, centrifuge the plate, seal it with a plate seal, and incubate at room temperature for 2 hours.

	Maximum valueComparison	sample	NC
Step one	2 μL 1× DetectionBuffer	2muL Gradient dilution of the sample to be tested	10uL 1× DetectionBuffer
	4 μL Tag1-IL17A proteinWorking fluid
	Incubate for 10 minutes.
Step two	4 μLTag2- IL17 RA proteinnWorking fluid
	10μLMix
Step three	Cover with a sealing film and incubate at room temperature for 2 hours.

3.Detection

Detect on a TR-FRET-compatible microplate reader..The excitation light is 320/340 nm, and detection is performed at 620 nm and 665 nm.postWavelength of the radiation.

[Result Calculation]

1)Calculate signal value(Ratio)The 665 nm fluorescence signal is multiplied by 10,000 compared to the 620 nm fluorescence signal..

Ratio = (665/620)times10000

2)Calculate based on the signal value.Net signal:

Net signal= (Std-NC)/NC×100

3)Calculate CV (%):

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

[Data Example]

The following data should not be used as a substitute for the data obtained in the experiment; it is provided for illustrative purposes only, and the results mayDue to the reading boardInstrumentIt varies from device to device.

Note: RecommendedMicroplate (384-well plate, white, flat-bottom)

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Unione® TR-FRET Human IL17A/IL17RA Binding Kit