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Description
Mouse TIMP4 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: 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: 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. 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. 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 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 Tissue Inhibitors of Metalloproteinase 4 (TIMP4) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Tissue Inhibitors of Metalloproteinase 4 (TIMP4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Tissue Inhibitors Of Metalloproteinase 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Matrix metalloproteinase inhibitor 4 is an enzyme encoded by the TIMP4 gene. This gene belongs to the tissue inhibitor of metalloproteinases gene family. Proteins encoded by this gene family are inhibitors of matrix metalloproteinases, a group of peptidases involved in extracellular matrix degradation. The secreted reticular protein encoded by this gene is involved in regulating platelet aggregation and recruitment and may play a role in hormonal regulation and endometrial tissue remodeling. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, and other biological fluids |
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4.8 ★★★★★
Based on 21 reviews
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Product Reviews
★★★★★ 5
Will definitely get more for my pig ear loving boy!
Flavor Name: Pig Ear, Size: 8 Count (Pack of 1)
My shar pei mix baby LOVES these! He is about 4 months old, teething and always has food on the brain. These do the trick! One ear lasts him about 2 days if he is really going at it, sometimes longer. He will work on it for hours at a time.
They have little to no smell. He chews them on his white dog bed and they leave no stains or marks. I love that there aren't any extra ingredients so I know exactly what he is ingesting.
I will DEFINITELY be buying more of these!
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Reviewed in the United States on June 16, 2025
★★★★★ 3
Is Milo allergic to pig ears?
Flavor Name: Pig Ear, Size: 10 Count (Pack of 1)
Milo sniffed the pig ear, paused, and then took it to his spot to eat it. A few minutes later he started rubbing his head on the floor. I also bought the cow ears and he ate them with no problem. I’ve snuck him the occasional piece of bacon so I don’t think he’s allergic to pork. I love and trust Nature Gnaws.
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Reviewed in the United States on May 28, 2025
★★★★★ 5
Great quality!
Flavor Name: Pig Ear, Size: 8 Count (Pack of 1)
I do not review items very often, but after considering that I have ordered these no less than 30 times, I decided it was time. I have four dogs who absolutely love them, so I go through them pretty quickly. They are generally very large, with occasionally one smaller one in the bag. These are the best price/quantity pigs ears I have been able to find. Each bag has eight ears. My dogs range in size from a (very food-motivated) mini dachshund to a pit bull, so the treats are good for any size. I order them for my sister’s and my boyfriend’s dogs, and my boyfriend breaks them in half to give to his dog if you don’t want them to have the whole one all at once. Would highly recommend!
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Reviewed in the United States on April 28, 2020
★★★★★ 5
Perfect toy for an active dog!
Color: Large size (7.9")
My dog absolutely loves this ball with handles! It’s quickly become one of his favorite toys, and he gets excited every time we bring it out to play.
The vibrant colors are a huge plus—they make it super easy to spot in the grass, which saves a lot of time during outdoor play sessions. The handles are great for interactive play too, especially for tugging and tossing.
I’m really impressed with how well it’s holding up. Even with constant pulling, tugging, and rough use, it’s remained durable and hasn’t shown any signs of wearing out.
Overall, I’m very happy with this purchase—and if my dog could write reviews, it would definitely get his paw stamp of approval! 🐾
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Reviewed in the United States on May 24, 2026
★★★★★ 5
Not for strong chewers!
Color: Large size (7.9")
I have an English Springer Spaniel who is 1.5 yrs. She had this torn apart within the first 5 minutes. This is not for strong chewers!
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Reviewed in the United States on February 25, 2026