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Description
Human CKLF1 ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect specimens using EDTA or heparin as anticoagulants and centrifuge them at 1000×g for 15 minutes at 2-8℃ within 30 minutes of collection. The supernatant can be tested or stored at -20℃ or -80℃, but repeated freezing and thawing should be avoided. 3. Tissue Homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove any 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 freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. 4. Pre-Assay Preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the gradient working solution of the standard: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration is 200 pg/mL). Then dilute to the following concentrations: 200 pg/mL, 100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, 6.25 pg/mL, 3.125 pg/mL, and 0 pg/mL. Serial dilution method: Take seven EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 200 pg/mL standard working solution into the first EP tube and mix thoroughly to make a 100 pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 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). 5. 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. 6. 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 uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate are sequentially added to microwells pre-coated with a capture antibody against Chemokine-like factor 1 (CKLF1). 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 Chemokine-like factor 1 (CKLF1) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Chemokine-like factor 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Chemokine-like factor 1 (CKLF1) is the first member of the MARVEL family of transmembrane domain proteins similar to CKLF to be defined and the most studied of its four isoforms. It acts through the CCR4 receptor on CD4+ Th2 lymphocytes, neutrophils, monocytes, macrophages, dendritic cells, and perhaps other cells bearing the CCR4 receptor. | |||||||||||||||||||||||||||||||||
| 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 | 3.12-200 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and tissue homogenates |
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4.1 ★★★★★
Based on 11 reviews
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Product Reviews
★★★★★ 4
Great toy, as long as dog is NOT ALLOWED to chew on it
Size: Large Triple
Let me start by saying that I like this dog toy. My big dog LOVES tugging with it. As long as you keep away when the dog is in a chewing mood, it lasts a long time (my 1st one lasted a year), however, it WILL NOT last if the dog chews on it, as I found out with this one. A friends dog chewed on it while she was behind a snow mound (mostly out of our sight) when it was only a week old. One end ring was barely holding together when we discovered her chewing it. Fortunately it was an end ring, so I still have 2 connected rings for tugging with.
It is a great tug toy! But it is also a TERRIBLE chew toy, so make sure to take it away when your dog starts to chew on it and it will give you long service.
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Reviewed in the United States on February 14, 2021
★★★★★ 5
Fabulous toy!!
Size: Large Triple
My GSD is always up for a good game of tug-o-war. I purchased these in large and I think I love them as much as my dog. They are super strong and hold up to his 100lb self pulling on one end and my “blah blah-lb” self pulling on the other. (Ha! You thought I would say how much I weigh) I even bought another pair for our cabin so we have some to play with at both places. He isn't a sit and chew on things kind of dog. He gets plenty of playtime and bully sticks for chewing. Not to mention that I wouldn't let him use them as a chew toy anyway. There are toys for play and toys for chew. Therefore, I cant say anything on how they hold up for chewers. So if you let your dog just sit and chew on it instead of giving them something designed for strong chewers, then no it might not hold up to that. All I can say is my German Shepherd is 100lbs with sharp teeth and stronger than me now and honestly he is probably stronger than I have ever been in my life, and they hold up to the pulling and tugging of both of us. Thats what they're designed for and they serve their purpose excellently.
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Reviewed in the United States on January 22, 2026
★★★★★ 1
Won't purchase this one again.
Size: Large Triple
J.W. used to make a ring toy such as this, that was softer & thicker with a smaller black ring in the middle, that our dogs all, absolutely loved!!!!! But this one totally bores them to the point none of the dogs will pay it any attention! Please bring back the original 3 ring toy, that J.W. has now changed to just the one ring! I feel like they must have discontinued the toy with the 3 rings they used to sell, with the smaller black ring in the middle? I won't waist my $ on another one of these! Sadly, none of our 3 dogs will play with this specific toy at all! & I generally prefer to purchase J.W. brand toys for our dogs. But this toy doesn't attract them in anyway. It just sits around.
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Reviewed in the United States on March 6, 2026
★★★★★ 3
Great idea but didn't hold up!
Size: Large
My dog and I really enjoyed this as a tug toy to use where it kept my hands away from accidental nips. When we initially got it we let her play solo with it after our tug session and I noticed that she left little teeth puncture holes so we packed it away for awhile and exclusively used the other new toy we bought for her. After a few weeks we took this back out and, again, we loved it and this time I allowed her to play with it longer and although it was showing a lot of the little tooth marks it seemed to be holding up so I left it out with her. None seemed deep, just seemed like impressions from her teeth.
We continued to enjoy it for about a week where she'd carry it over to us for tug sessions, loop it on our feet when we were sitting watching tv and chewing on it a bit. I'd look at it when we'd tug and, like I said above, it seemed to be holding up. After one session of tug when she took it off to chew on and I was watching some tv I checked on her after awhile and she had bitten through the black side and had chewed up into small pieces about a two inch section of it. Small, little hard bits were in a pile on the floor. She didn't just bite through, it had broken into small, little pieces and hopefully I was able to get all of them and she didn't swallow any!
So if you want to get this exclusively as just a tug toy and not something for your dog to chew on and plan on putting it away after I think it will be okay for that and should last but if your dog is a serious chewer like ours is you may not want them to be allowed to chew on this. Seemed like it was going to last and then suddenly I was throwing away half of it and hoping she didn't eat any of the black bits. We still have the yellow side and will take that out for some tugging but no longer for her to play with freely!! She did tremendously love running around with this thing and would flip up the loose end and hook it on her face or our feet and was so cute to watch her!! Wish it had lasted!!
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Reviewed in the United States on May 9, 2013
★★★★★ 5
Name holds true.
Size: Large Triple
Just bought my second set of these.. first set lasted my healer almost 4 years and are still together but starting to split at the smaller seams so you can’t play as aggressive as my dog would like. We bought the second set and now she has her second wind! She loves tugging and I love knowing this toy will last a good while especially with an active dog!
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Reviewed in the United States on January 4, 2025