TLR4 ELISA 试剂盒

• Pre-coated, ready to use 96-well strip plate
• Plate sealer for 96 wells
• Standard
• Diluents buffer
• Detection Reagent A
• Detection Reagent B
• TMB Substrate
• Stop Solution
• Wash Buffer (30 x concentrate)
• Instruction manual

• Microplate reader with 450 ± 10nm filter.
• Precision single or multi-channel pipettes and disposable tips.
• Eppendorf Tubes for diluting samples.
• Deionized or distilled water.
• Absorbent paper for blotting the microtiter plate.
• Container for Wash Solution.

• Adherent cells should be detached with trypsin and then collected by centrifugation (suspension cells can be collected by centrifugation directly).
• Wash cells three times in cold PBS.
• Resuspend cells in PBS (1x) and the cells was subject to ultrasonication for 4 times (or Freeze cells at≤-20 °C. Thaw cells with gentle mixing. Repeat the freeze/thaw cycle for 3 times.)
• Centrifuge at 1500xg for 10 minutes at 2 - 8 °C to remove cellular debris. Cell culture supernates and Other biological fluids - Centrifuge samples for 20 minutes at 1000xg. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C. Avoid repeated freeze/thaw cycles.

1. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance.
2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary.
4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals.
5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products.
6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit.
7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results.

1. Prepare all reagents, samples and standards.
2. Add 50μL standard or sample to each well. And then add 50μL prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37 °C.
3. Aspirate and wash 3 times#Add 100μL prepared Detection Reagent B. Incubate 1 hour at 37 °C.
4. Aspirate and wash 5 times.
5. Add 90μL Substrate Solution. Incubate 15-25 minutes at 37 °C.
6. Add 50μL Stop Solution. Read at 450 nm immediately.

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Toll-like receptor 4 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 1.3. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Important note:
1. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit
2. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available.
3. Kits from different batches may be a little different in detection range, sensitivity and color developing time.Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for information.
4. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results.
5. Do not remove microtiter plate from the storage bag until needed.
6. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement.
7. Use fresh disposable pipette tips for each transfer to avoid contamination.
8. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer.
9. Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.
10. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too.
11. Kits from different manufacturers for the same item might produce different results, since we haven’t compared our products with other manufacturers.
12. The instruction manual also suit for the kit of 48T, but all reagents of 48T kit is reduced by half.
13. Valid period: six months.