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Description
Human TRAF5 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. 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 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/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 utilizes 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 TNF Receptor Associated Factor 5 (TRAF5) 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 TNF Receptor Associated Factor 5 (TRAF5) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human TNF Receptor Associated Factor 5 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | TNF receptor-associated factor 5 (TRAF5), also known as RING finger protein 84 (RNF84), is a protein encoded by the TRAF5 gene. This gene encodes a scaffold protein that is a member of the tumor necrosis factor receptor-associated factor (TRAF) protein family and contains an alphaprotein and TRAF homology (MATH) domain, a RING-type zinc finger, and two TRAF-type zinc fingers. TRAF proteins associate with members of the TNF receptor superfamily and mediate their signaling. This protein is a component of a multiprotein complex that binds to the cytoplasmic domain of the tumor necrosis factor (TNF) receptor and mediates TNF-induced activation. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates and other biological fluids |
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4.3 ★★★★★
Based on 7 reviews
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Product Reviews
★★★★★ 5
This worm, pretty sure it’s a worm and not a snake, can take a lot of abuse.
Size: X-Large (Pack of 1), Color: Blue Snake, Size: X-Large (Pack of 1), Color: Blue Snake
Snake, worm, whatever, my dog doesn’t care, she loves it all the same. My little friend can be kinda rough with her toys. This is no exception. The pictures are from roughly one month of giving it to her and include playing with it at least every other day or so. The top is holding up well, but the bottom has taken some damage, and two of the squeakers have long since been destroyed. Despite this, my dog still loves playing with it. We normally play tug of war together, but on her own she chews at it to get to the squeakers inside, which she promptly destroys, which I’m totally fine with, as quiet toys are much preferred, by me, not her. I believe there are 4 in the XL size that we got. We’ve had a few of these in the past. We tend to get about 6 months out of them. After she “lovingly” removes the squeakers, she doesn’t chew on it much, and we just use it for tug of war, but at that point she’s done enough damage to the underside that its time is limited. But, as it’s pretty low priced, for the time and enjoyment we do get out of it, it’s very much worth it. I do need to clarify, the durability is actually quite good, I don’t fault the product as at all, it’s just my dog is equally as good with destruction, but it should be noted this toy does last much longer than most of her victims. The color, (we got the blue and green) is also very vivid and nice, as is the texture. We’ll be purchasing again.
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Reviewed in the United States on September 23, 2024
★★★★★ 5
Awesome toy!
Size: X-Large (Pack of 1), Color: Blue Snake
My pitbull Stella loves these! Ive bought her two of the smaller snake toys in the past and she Absolutly loves them! The first one lasted about 6 months which is a lifetime compared to how long her other toys last lol and the second one lasted about the same. This time I decided to get the XL version. It has 6 squeakers instead of 3 like the smaller versions. I picked the teal and purple colors and it's like 43 inches long I think. Its the same length as her and shes a large size dog. She loves this thing! Its so cute watching her squeak each of the squeakers with her nose.
While this is a great toy it is not chew proof. In fact its quite easy for an experienced chewer to get into this toy. It is relatively strong but it wont stand a chance with a power chewer. Like I said this is the third one of these I have bought in roughly a year. They do last longer then more soft toys I will give them that. This is the only soft toy she has not destroyed in 5 mins. But after a week or so at least one squeaker will be missing.
For the average dog one of these snake toys will last a really long time and they are fun to play with. We play tug of war and fetch with it. Stella also loves when I squeak the toy she will squeak it back and we'll go back and forth for hours lol. One last thing it is loud! If noisy toys bug you, don't get this. The sqeakers are industrial strength lol
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Reviewed in the United States on February 7, 2021
★★★★★ 4
Durable toy but definitely NOT invincible
Size: Medium (Pack of 1), Color: Orange Gecko
BACKGROUND
I have two labradors and, with my friends dogs over, we have a combined combination of five labradors at a time. If you have seen the film "Marley & Me" then you will understand our dogs. They tear through concrete, dry wall, and, well, just about anything they can get their paws and mouths on. It is very important to have plenty of toys for them to keep pre-occupied as most of the destruction is caused by dogs that are bored.
Unfortunately, many items are not able to withstand this combined destructive force. I spotted these "Invincible" line of toys and thought they were worth a try. I ended up ordering basically one of every style and size.
APPEARANCE
The toys come in different sizes, colors, and shapes. The most common are the Gecko and the Snake. The toys are covered with a more durable cloth that is fuzzy and suppose to withstand quite a bit of chewing. Inside the toys are what are described as durable squeakers that, if punctured, will still squeak even after hours of being bitten and chewed upon. The larger the toy ordered, the more squeakers that are sewn into the toy.
RELIABILITY
I have to admit these toys did last about ten times as long as any other toy I have given my dogs. Unfortunately, nothing is indestructible. My dogs managed to find kinks in the lining of the toys that, once penetrated, will cause the toys to open leaving stuffing and squeak toys all over the place. The squeakers did withstand quite a bit of chewing before the squeaking went silent.
I noticed that the snake toy did last quite a bit longer than the gecko or the little dragon characters. The snake had separate compartments for the squeakers and so, if one was torn out, the others survived. On the Gecko once its middle section was torn open the squeakers were lost.
CUSTOMER SUPPORT
Not utilized
PROS:
++ Lasted about 10X times the length of time as other "invincible" toys
CONS:
++ They are not truly "invincible" and will be destroyed over time
CONCLUSION
The invincible line of toys did last a lot longer than most toys that I have purchased for my labradors. I would recommend the snake over the gecko or other styles. I have had to toss the geckos as they were shredded but the snakes are still with me to this day six months later. Also note, even though "Invincible" is in the title, these toys are definitely not although you will save an incredible amount purchasing these toys once and having a gradual destruction process versus having to buy new toys continuously over the same period of time that these lasted.
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Reviewed in the United States on October 18, 2012
★★★★★ 3
The toy is no match for my dogs.
Size: Large (Pack of 1), Color: Pink Snake
The toy itself is well made and the squeaker work well. Unfortunately, it only lasted about 2 weeks before my Treeing Walker Coonhound pup (super chewer) had it shredded. My Plott hound ( heavy chewer) just loved these! She is older and they last longer for her. However she does love to go for the seems first then rips out the squeaker. But she will carry it around for months like that. I think she like the material. I'll probably go back to Walmart for the smaller cheaper ones for now.
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Reviewed in the United States on April 8, 2026
★★★★★ 5
Durable and he loves it! It is Ryker Der Hauswolf approved and I recommend it.
Size: X-Large (Pack of 1), Color: Blue Snake, Size: X-Large (Pack of 1), Color: Blue Snake
Review of Outward Hound Durablez Snake – Squeaky Plush Dog Toy, No Stuffing for Less Mess, Interactive & Tough, 6 Jumbo Squeakers, Puppies & Dogs, X-Large, 40"
My best friend brought her 16-month-old German Shepherd up for a visit and I happen to love him to pieces! A few months ago, I bought the smaller version of this same toy snake for him and he LOVED it. Many squeaker toys he has eventually torn up, as puppies and others do. The smaller only has a small tear in the butt-end from playing some minor tug of war with it. This larger version is made just as well and with the same materials. It is very durable, but there are no toys indestructible. The only difference is the length, and it has 6 JUMBO squeakers as opposed to 5 smaller ones. It got here on the day of this post and he has not stopped playing with it! Now if you’re one that squeakers get on your nerves, this is not for you. But, if you’re like me and truly enjoy watching (and hearing) a fur baby having so much fun, then buy this now! The price is very reasonable in my humble opinion. It is Ryker Der Hauswolf approved and I recommend it.
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Reviewed in the United States on April 24, 2026