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Description
Human TBK1 ELISA KitProduct Specification Usage Required experimental equipment: 1. Microplate reader (450nm) 2. High precision pipettes and pipette tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre chilled PBS (0. 01M, pH 7. 4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and
Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. Cell Lysis Buffer: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit utilizes a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a TANK Binding Kinase 1 (TBK1) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of TANK Binding Kinase 1 (TBK1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human TANK Binding Kinase 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | TANK-binding kinase 1, also known as TBK1, is an enzyme encoded by the TBK1 gene. It is an enzyme with kinase activity. Specifically, it is a serine/threonine protein kinase. This kinase is primarily known for its role in innate immune antiviral responses. However, it also regulates cell proliferation, apoptosis, autophagy, and anti-tumor immunity. Inadequate regulation of its activity can lead to autoimmunity, neurodegenerative diseases, or tumorigenesis. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.7 ★★★★★
Based on 2032 reviews
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Product Reviews
★★★★★ 4
Awesome, but not the best squeakers.
Color: Purple
I bought the orange gecko for my 5-month-old Corgi puppy. He absolutely loves it. He came up with a "game" (sort of) where if I pick it up and start squeaking one of the ends/squeakers really quickly over and over, he'll grab the other end/squeaker and start doing the same thing with me. It was really cute until one of the squeakers stopped working about a week after I bought it... but I guess the toy wasn't too expensive, and he doesn't really seem to mind that one of the squeakers doesn't work anymore. I might buy another next time I do an Amazon order (so I can get the free shipping) and see if it lasts a little longer.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on August 2, 2013
★★★★★ 5
Great Toy For a Great Price
Color: Purple
My Aussie loves toys and plays with them as though she is a small child. She has a basket and will go to the basket and dig through it until she finds what she is looking for. Lately she has been using the bungee gecko as her toy of choice. She gets bored easily and will rip the stuffing out of most toys, the fact that this one has lasted 6 weeks or so is impressive with no rips, holes or missing limbs. Her favorite way to play is to come over to me and nudge me to play tug of war and we have broke many toys this way but so far we can play tug a war several minutes at a time and the gecko is still in one piece. For the price which is considerable less than some of her other toys I have already ordered a couple of replacements for the future. This is a great well built dog toy that has has a lot of give and can be stretched without ripping.
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Reviewed in the United States on May 3, 2013
★★★★★ 3
No longer as durable
Color: Purple
I have one of these that has made it through 2 dogs but now the bungee is stretched out. It's been such a well loved toy I recently got another one. They've changed the plush fabric. The old one was indestructible. This new one is much thinner and easily torn. I wish they would go back to the old plush fabric.
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Reviewed in the United States on February 4, 2024
★★★★★ 5
Great Toy!
Color: Orange
This has been a great toy for our 11 week old German Shepherd. She absolutely loves it. I can get out all of her toys and lay them out with the exception of her "Lizzy" and she will immediately go to her toy bin and get it herself. Needless to say it is her favorite toy. She has had it a week now and she plays with it a lot, chewing and squeaking. It's a pretty amazing toy to have held up this long already and to date shows no sign of wear or damage. Lizzy just keeps going and going.
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Reviewed in the United States on April 4, 2014
★★★★★ 1
This item came with plush missingand thread unraveling on one leg
Color: Blue
I bought this toy because my dog spent a week playing with the same toy from Zanies that belonged to a visiting dog. My dog was very gentle with this toy, which he loved, so we decided to find one for him. The toy that arrived had no Zanies label and missing plush on the bottom where the label should have been, plus other bare spots in the area that showed the white mesh beneath. It also had a long thread at the joint of a rear leg so I knotted it at the joint and clipped it to prevent my dog from pulling at it but it's apparent that the sewing is loose and wont last long. The color was also much lighter than the toy he'd been playing with that belonged to the guest dog but I figured it might just be from a different dye lot (it was not bought at Amazon but their local pet store.) I am not convinced this is a real Zanies toy. There were two labels in French and it said it was made in China. I am returning this toy and will get a replacement and see how it looks. If it seems like it will fall apart or is not the real thing I will return it immediately. If it has a label and is in good shape and sewn well at the seams I'll revise this review.
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Reviewed in the United States on August 4, 2020
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