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Description
Rat TGF-a 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 the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold PBS and resuspend 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 1000pg/mL). Then dilute to the following concentrations: 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 1000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500pg/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). 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 Transforming Growth Factor Alpha (TGF-a) 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 level of Transforming Growth Factor Alpha (TGF-a) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Transforming Growth Factor Alpha ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Transforming growth factor alpha (TFG-α) is a protein encoded by the TGFA gene. As a member of the epidermal growth factor (EGF) family, it is a mitogenic polypeptide. It can induce epithelial development and transform a variety of cells. Its amino acid sequence is similar to that of EGF. It is produced by macrophages, brain cells, and epidermal cells. Similar to EGF, it acts by binding to the cell surface receptor EGFR. When bound to the receptor, which mediates protein kinase activity for cell signaling, the protein is activated. It is a transforming growth factor and a ligand for the EGFR, activating signaling pathways involved in cell proliferation, differentiation, and development. This protein can act as a transmembrane-bound ligand or a soluble ligand. This gene has been implicated in many types of cancer and may also be involved in some cases of cleft lip/palate. In the stomach, it is produced in the normal gastric mucosa and has been shown to inhibit gastric acid secretion. It can also be produced in macrophages, brain cells, and keratinocytes. It induces epithelial development. | |||||||||||||||||||||||||||||||||
| 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 | 15.62-1000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.6 ★★★★★
Based on 17 reviews
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Product Reviews
★★★★★ 5
Best toy for heavy chewers
Color: Green
My dog is an aggressive chewer and I have trouble finding toys that last longer than 5 minutes before being so destroyed it is no longer safe to let him keep it. He loves this toy and naws on it for hours daily. Yes, it has multiple chew marks, but it is still intact completely. No little pieces have been chewed off. I highly recommend this toy for any dog especially those who destroy every toy in seconds like mine does.
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Reviewed in the United States on October 12, 2025
★★★★★ 2
NOT Indestructible and a Nylon Product (NOT A CHEW TOY)
Color: Green, Color: Green
I gave this dog toy 2-stars based on my dog toy rating system (DTRS) for “Bruce” the Green Alligator!
Unfortunately, I didn’t notice in the fine print of the product description on the website that this toy consisted of Nylon. If I noticed that description, I would’ve never purchased this product. Please do your own research on Nylon wrt teeth. I wasn’t impressed with the toy construction out of the box. The nylon head and tail sandwich the rubber body with soft rubber spines on Bruce’s back for easy gripping. This is not an “Indestructible” dog toy.
Safety (1-rating):
1. I was always concerned about my dog’s teeth possibly cracking while he was chewing on Bruce’s Nylon ends. Thankfully, his teeth were fine throughout the use of this toy.
2. The Nylon came off in small pieces after a few hours and continued for the life of the toy; see picture. I never intended for my dog to swallow pieces of Nylon.
3. Bruce’s rubber rear end is where most of the damage occurred, as my dog was tearing off pieces of rubber and spines, which he thankfully passed through his system over a period of 2-3 days after I threw Bruce away; see picture.
4. The chewed-up Nylon ends were very hard, rough, sharp and pointy. There were occasions where I noticed my dog’s gums were bleeding as a result of chewing this toy.
5. Bruce is big enough not to swallow in its entirety, which is why I’m giving this a 1-rating for safety.
6. This is a molded product and not a plush product.
Durability (2-rating):
1. It took my dog 1 to 2-weeks to get the toy in the shape that is shown on the posted photos.
2. The Nylon was immediately being scratched and scraped apart on day-1. Then I noticed pieces of Nylon coming off of Bruce after 3-4 days.
3. The rubber was immediately being punctured with small divots around the body on day-1. I noticed over the next couple of days that my dog was focusing on the rear end and trying to pull the rubber off the Nylon tail.
4. The rubber started coming off more quickly after 1-week, until I removed it all together.
Squeaker (N/A):
1. Bruce has no squeaker.
Fatality (2-rating):
1. Bruce had a longer life than most of my dog’s toys, but then again, I expected this to last much longer as the seller claims it is “Indestructible”, which it is not.
2. I tried filing off some of Bruce’s chewed up Nylon ends to smooth them out for more chew time, but came to my senses and just threw him away for safety concerns only.
In conclusion, “Bruce” the Green Alligator should not be given as a chew toy, but more as a fetch toy, unless you want your dog to digest the Nylon and rubber materials.
Days of Enjoyment
Given to Dog: 12/16/2024
Dog Toy Death: 01/08/2025
Total Days Used: 13 days (11 days spent away from home during the holidays)
About my dog “Cannoli” for comparison
Breed: Blue Healer / Lab mix
Sex: Male
Age: 8-months
Weight: 45-lbs
Size: Medium
Energy Level: High
Chew Rating: Aggressive
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Reviewed in the United States on February 26, 2025
★★★★★ 5
This has been a challenge for my extreme chewer! The toy is winning
Color: Green
Indestructible? Not for my pitbull. Except on this toy. She has slowly chewed some of the outer portion on the backside, but it still has a lot of gnawing time left. This is her go-to toy now. she carries it nearly everywhere. She loves it. I am amazed at how well it is holding up. Within minutes, hours or a few days, the other "indestructible" toys I've purchased, never survived as described.
This is a heavy toy! Not for the weak! I cannot stress enough the extreme chewer my dog is. Yet, I can see this toy lasting months. It won't be pretty then but it will most likely still be a favorite chew toy. I'm glad I found this and will be buying more.
FYI's... 1) Chewability-5 stars 2) Softness-1 star, it's solid but chewable, 3) Bounce-0 stars, No bounce, it's heavy!, Playability- 5 stars. My dog plays with it, lets it lay with her when resting. Nearly always within her reach. She sleeps with it! That's love.
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Reviewed in the United States on January 2, 2026
★★★★★ 5
My buddy has been chewing on this for an hour!
Color: Green, Color: Green
This dog toy has been a great addition to our dog’s toy collection! My dog can be a pretty aggressive chewer, so I’m always looking for toys that are durable and can actually hold up to daily play. This one has been surprisingly tough and has lasted much longer than many other toys we’ve tried.
The material feels strong and well made, and the design makes it easy for my dog to grip and chew. He loves carrying it around and tossing it during playtime. It’s also great for keeping him busy and helping reduce boredom, especially when he has extra energy to burn.
Another thing I like is the texture of the toy. It seems to help keep my dog engaged, and it’s sturdy enough that I don’t worry about it falling apart right away like some softer toys do. It’s been used for chewing, tugging, and even some fetch around the house, and it’s still holding up really well.
The only small drawback is that I wish it had a squeaker inside. My dog absolutely loves squeaky toys, and I think that would make it even more exciting and keep him interested for longer periods of time.
Overall though, this is a very solid and durable dog toy that’s perfect for dogs that love to chew. If you’re looking for something tough that can help keep your dog entertained and reduce boredom, this is definitely worth try
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Reviewed in the United States on March 12, 2026
★★★★★ 5
Great Toy For Aggressive Chewers
Color: Green, Color: Green
My Boy loves it! It is a little heavy but it keeps him busy. Very strong and sturdy for aggressive chewing. We named it Ally the Alligator. He loves it definitely would recommend!
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Reviewed in the United States on May 25, 2026