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Description
Human VAChT 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 uses 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 vesicular acetylcholine transporter (VAChT) 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 vesicular acetylcholine transporter (VAChT) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Vesicular Acetylcholine Transporter ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The vesicular acetylcholine transporter (VAChT), also known as solute carrier family 18, member 3 (SLC18A3), is encoded by the SLC18A3 gene, located within the first intron of the choline acetyltransferase gene. It is a neurotransmitter transporter responsible for loading acetylcholine into the secretory organelles of neurons, enabling its secretion. It acts as an antiporter by transporting ACh into vesicles through the exchange of protons (H+) previously pumped into the vesicles for diffusion. ACh molecules are then carried into the vesicles by the action of exiting protons. PET imaging of this transporter may provide insights into the early diagnosis of Alzheimer's disease. | |||||||||||||||||||||||||||||||||
| 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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4.3 ★★★★★
Based on 957 reviews
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Product Reviews
★★★★★ 4
Doesn't really taste like bacon. I know becaused I chewed it. Great as a slower eating thingy.
Style: Dental Chew, Flavor Name: REAL Bacon, Size: Dental Medium
So my dog has this thing where she does not want to chew on anything that she is supposed to chew on unless it's a knuckle bone that she cannot be trusted with (because she bites off fragments of bone) or one of those flexible, edible nylabones for "gentle" chewers, which she will eat in a single sitting. If I want to get her to chew something that might actually withstand her for more than an hour, I have to pretend it's mine. It's a secret forbidden treat that I love very much and just "accidentally" left within her reach to steal and covet.
So it's pretty much standard practice at this point that, when I get her a new chew toy, I gotta carry it around for a while, pretending to chew on it. I got this bone in the mail and did exactly that, but I noticed right off the bat that, for a bacon-flavored thing, it didn't smell like anything at all. It was just this plain sort of plastic scent. And that's how I ended up actually chewing on it instead of just miming it out because maybe the bacon flavor was under the initial layer? But no, it wasn't. It was just bacon-colored plastic. So if your dog is the type of chewer that will chew regardless of flavor, this should work out fine. If not, go for something else. My dog did not give a heck about this bone, even once the "this is actually mine but you cleverly stole it away" ritual was completed.
After that, I tried putting it in her bowl with her food, thinking maybe the flavor of the food would rub off enough that she'd want to chew it. She didn't, but what did happen is the reason I'm giving this a positive review at all. While it fails utterly as a chew, it makes a really great impromptu "stop eating so fast, you tornado shaped like a dog" mechanism. We've bought actual slower eating bowls for her and never had any success, because she just gives up and cries if she can't get her face into the crevices. But when we put this in her regular round bowl and pour her wet food around it, she's able to nose the benebone around in the bowl as she eats her food, but it does slow her down considerably. She's way less farty and whiny after food time, which anyone who has experienced how specifically racid dog farts can be will know is a blessing. So, thanks, Benebone. For us, your product is worthless as a chew, but invaluable as a makeshift slower eating thingy.
Edit: Benebone got in contact with me about my review and offered to refund my purchase and send me some different products to try. I declined the refund, because I do find the bone I bought useful for the above-described purpose of slowing my dog down when she eats. But I did accept the samples. She's still pretty unimpressed with them, and I will probably end up passing them along to other dog-havers I know, but I myself am pretty impressed with Benebone's customer service and so have changed my review from 3 stars to 4.
It was "suggested" that I mention that other dogs will probably be more satisfied with Benebone's flavors if they are not as picky as my dog is, and that sampling a Benebone yourself is maybe not the most reliable way to test its tastiness, when dogs have much more sensitive noses/tongues than humans. Which is certainly true! But I don't think my dog is a complete alien for wanting something with slightly more discernible flavor and scent. And I do think some of the advertising for Benebone implies more of that than you actually get in the finished product. So I'm gonna have to double down on my previous assessment that, if your dog likes to chew for the sake of chewing, Benebone will probably really appeal to them. But if they're a more food-motivated chewer, you're better off with fully-edible chews like rawhide or bully sticks.
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Reviewed in the United States on August 27, 2018
★★★★★ 5
My Dog’s Favorite – Durable & Keeps Them Busy
Style: Fishbone, Flavor Name: REAL Jack Salmon, Size: Small
This is my dog’s favorite chew by far. I used to go through chew toys constantly and could easily be replacing them every week, but this one actually holds up.
It keeps my dog engaged and gives me peace of mind knowing it’s something they enjoy and I can trust in terms of durability.
If you have a dog that goes through toys quickly, this is definitely worth trying.
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Reviewed in the United States on April 26, 2026
★★★★★ 5
My Pitbull absolutely loves the bacon flavor.
Style: Stick, Flavor Name: REAL Bacon, Size: Medium
This is officially my dog’s new favorite toy. She plays and chews on this thing nonstop. She has dozens of toys and she doesn’t touch any of them unless, she forgets where she left her bacon stick. This is a must for the perfect Dental Toy.
I kinda suspected she’d love these things, so I also bought the knob in chicken flavor. She likes the chicken but, it’s so obvious she loves the bacon flavor. Smart girl. I absolutely love bacon also, bacon is perfect by itself, and it makes everything else much better. Except a bacon martini, so disgusting.
The main reason I bought two was to put one in my vehicle, for road trips. My dog is my child, my Princess, so she goes almost everywhere with me. I knew this would be great for keeping her entertained and distracted while we’re out running errands and stuff. I always keep separate toys in the cars for her to chew on. A few years ago I forgot to grab a few toys out of the bin and she decided that boredom was a good excuse to chew on a seatbelt, no cool. Ever since then I decided to have dedicated toys that stay in the vehicles, so I’ll never forget again. The toys she loves the most are the ones I buy multiples of. One for the house and one for each vehicle. For me it’s simpler.
I did notice at first, her gums did bleed a tiny bit. She’s a Pitbull, very aggressive chewer but, only with certain toys. This is definitely one of those toys. The bleeding stopped after a few days and things are back to normal.
This is truly a unique product. I call it a toy but it has many positive perks. It’s her favorite. She plays with it all the time. Any time I need to distract her for something I grab the Benebone and show it to her, she immediately gets excited and self soothes. I feel like dental hygiene is the biggest bonus of all though. I’d say 80% of her toys have dental hygiene in mind, that’s very important to me, along with not having bad breath. She see’s a flavored toy, and I see her getting dental praises at every vet visit. All parties are extremely happy.
This toy is also extremely durable. With that said, I knew she wouldn’t be able to chew through it overnight, or even in a week. It’s tough finding toys that she won’t chew through overnight. This product is lasting her a long while. It’s not always about the price. To me, a toy lasting more than a few months is more important than the money spent. Based on the video ad I saw online I knew it’d be a durable product, plus I love supporting happy families that bring something different, and unique to market. Happy family equals happy products. Once it arrived and I could feel the material and texture in my hands I knew for sure “no way she’s gonna chew through this fast, or maybe not at all”. Even though it really is lasting for a long time, I still feel like she’s chewing through it faster than other dogs. That’s just my particular dog.
I had a tough time picking the right size for my dog. My Pitbull is small, about 35lbs. But man does she chew in the 100lbs + size range. I really wanted the biggest size for her but, I thought I don’t want it so big she won’t be able to even chew on it, or even just getting her jaws around it. So I went with ONE SIZE ABOVE the size/weight chart that is provided in the description, and it turned out to be perfect for her. It’s small enough she can chew and have fun. It’s too big for her to bite chunks off. In the past she’s bitten chunks off all her small toys.
I put both the chicken and bacon in front of her to see which one she’d grab first. Of course it was the bacon. Then it took them both and put them in her toy bin to see which she’d grab. Again the bacon right away. I love bacon too, great choice thinking like me. So I the bacon flavor stays in the house, and the chicken I put in my car for road trips.
The noises my dog makes while chewing and playing with this thing drives my wife nuts. It doesn’t bother me at all, not even a little. Thanks to multiple tours to Iraq, I can completely ignore petty noises and sounds. My wife on the other hand, will tell her to play in a different room. She listens and goes into a different room. That applies to both us for all of our dog’s toys, not just this one. To be honest these Benebones do have kind of a hard texturized plastic feel to them, so yes it does make a tiny bit of noise when she’s playing and chewing.
I can almost promise and guarantee that any every dog will love the Benebone in any of the shapes, sizes, and flavors. It really is the perfect toy for a dog. Especially for aggressive chewers like my Pitbull, Princess Polly. She keeps her favorite toys in her bed. This Benebone has a spot beside her big squeaky tennis ball, rubber tug-a-war, and her deer antler. I haven’t even seen her touch the other toys since I bought the Benebone.
I am extremely happy with my purchase and will for sure be buying more forever. I recommend buying multiple for inside your home and for each vehicle. You will not be disappointed, unless you buy one and your dog looses it and forgets where they hid it for later.
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Reviewed in the United States on March 30, 2021
★★★★★ 5
Great for heavy chewers
Style: Tripe, Flavor Name: REAL Beef, Size: Large 1-Pack
It lasts a long time. I have heavy chewers. And this is going strong for months now. It’s their new go to toy. I have americanbulldogs Scott type. 88lbs female and over 100lbs male.
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Reviewed in the United States on May 10, 2026
★★★★★ 5
🐾Ridiculous aggressive chewer🐶
Style: Stick, Flavor Name: REAL Bacon, Size: Medium
Sooooo happy I finally found something that my moms pup couldn't chew up! Not going to like its been very tough. She has now for at least a week (sorry days pushing together 🤪)
Let's just say the pup is a chihuahua mix that LOVES to chew. Emphasis on the chewing youre lucky if a toy lasts more than a day let alone a week 🤣
This is so durable I may not need one for her for a while but this will come HIGHLY recommended for ridiculously aggressive chewers.
Suitable for ang size dog just make sure you go one size bigger then you think you should. Quick easy chew toys but definitely has some weight to it.
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Reviewed in the United States on May 18, 2026