Product Name:	Human TIMELESS ColorStep ELISA Kit
SKU:	AEFI02685
Size:	96T
Reactivity:	Human
Range:	0.156-10ng/ml
Sensitivity:	0.094ng/ml
Detection Method:	Sandwich ELISA, Double Antibody
Detection Wavelength:	OD450
Reaction Duration:	120 minutes
Synonyms:	Protein timeless homolog ELISA Kit, hTIM ELISA Kit, TIMELESS ELISA Kit, TIM ELISA Kit, TIM1 ELISA Kit, TIMELESS1 ELISA Kit

Kit Components:

Components	Quantity		Storage
	48T	96T
ELISA Microplate(Dismountable)	8×6	8×12	Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
Lyophilized Standard	1vial	2vial	Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
Cap/Det Ab (Ready to use, blue)	3ml	6ml	2-8°C (Avoid Direct Light)
HRP-Streptavidin (Ready to use, orange)	5ml	10ml
TMB Substrate	5ml	10ml
Sample Dilution Buffer	20ml	20ml	2-8°C
Stop Solution	5ml	5ml
Wash Buffer(25X)	15ml	30ml
Plate Sealer	3 pieces	5 pieces
Manual	1 copy	1 copy

Other Materials Required (Not provided)

Microplate reader (wavelength: 450nm)
37°C incubator (CO2 incubator for cell culture is not recommenced.)
Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
Sterile tubes and Eppendorf tubes with disposable tips
Absorbent paper and loading slot
Deionized or distilled water

This kit was based on sandwich ELISA method. The experiment lasted 120 minutes. Capture antibody was conjugated to an affinity tag that was recognized by a specific antibody coated on the QuickTest plate. Add the Cap/Det Ab working solution into each well, then add the standards and pilot samples into individual wells. If the sample contains TIMELESS, a capture antibody-TIMELESS-biotin-detection antibody complex was formed. After incubation, unbound conjugates were removed by wash buffer. HRP-Streptavidin was added. After washing, TMB substrates were added to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. The concentration of TIMELESS in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.

Recovery:

Add a certain amount of TIMELESS into the sample. Calculate the recovery by comparing the measured value with the expected amount of TIMELESS in the sample.

Sample Type	Recovery Range(%)	Average(%)
Serum(n=5)	92-104	96
EDTA Plasma(n=5)	92-100	97
Heparin Plasma(n=5)	90-105	95

Linearity:

Dilute the sample with a certain amount of TIMELESS at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
Serum(n=5)	86-103%	98-104%	84-100%
EDTA Plasma(n=5)	91-101%	85-103%	83-99%
Heparin Plasma(n=5)	92-103%	86-103%	85-101%

Precision:

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.

Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Item	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/ml)	0.31	1.18	5.28	0.29	1.21	5.09
Standard deviation	0.01	0.04	0.18	0.01	0.05	0.23
CV(%)	4.45	3.71	3.46	5.08	4.35	4.6

*Note:The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Protocol
1.	Take out the required plate wells, add 50ul Cap/Det Ab into each well, then add 50ul Standard or Sample into individual well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure thorough mixing then static incubate for 60 minutes at 37°C.
2.	Washing: Wash the plate twice without immersion.
3.	Add 100ul HRP-Streptavidin (orange) into each well, seal the plate and static incubate for 30 minutes at 37°C.
4.	Washing: Wash the plate five times without immersion.
5.	Add 90ul TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
6.	Add 50ul stop solution. Read at 450nm immediately and calculate.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay..
Plasma	EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline..
Tissue Sample	Generally tissue samples are required to be made into homogenization. Protocol is as below: 3.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. 3.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF). 3.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. 3.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. 3.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again. Notes:PBS buffer or the mild RIPA lysis can be used as lysates While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing.
Cell Culture Supernatant	Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..
Cell Lysate	5.1. Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre- cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. 5.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3- 5mm probe, 150-300W, 3-5s/time, 30s intervals for 1-2s working). 5.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at - 80°C for future’s assay. Notes:Read notes in tissue sample Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml.
Other Biological Sample	Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay..

UniProt ID:	Q9UNS1
Sample Type:	Serum, Plasma, Cell culture supernatant, Cell lysate or tissue lysate, Other biological fluid samples
Storage:	2-8°C(Sealed), Don't cryopreserve.
Specificity:	Specifically binds with TIMELESS , no obvious cross reaction with other analogues.