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Description
Mouse bACE1 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 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. 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. 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. 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. 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 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 Beta-Site APP Cleaving Enzyme 1 (bACE1) 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 Beta-Site APP Cleaving Enzyme 1 (bACE1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Beta-Site APP Cleaving Enzyme 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | β-Secretase 1, also known as β-site amyloid precursor protein cleaving enzyme 1, β-site APP cleaving enzyme 1 (BACE1), membrane-associated aspartic protease 2, memapsin-2, aspartic protease 2, and ASP2, is an enzyme encoded by the BACE1 gene. BACE1 is an aspartic protease that is important for the formation of myelin sheaths in peripheral nerve cells: in mice, BACE1 expression is high during the postnatal period, when myelination occurs. | |||||||||||||||||||||||||||||||||
| 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 homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.9 ★★★★★
Based on 27 reviews
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Product Reviews
★★★★★ 3
Can't choose the dino... sad!
I have a beagle/jack russel mix. Needless to say, he is very high-energy and does best when given a job to do that involves chewing.
He LOVES his Novabone and Nylabone- but tears thru the larger sizes in a single afternoon- so when I saw this I was very happy, because it is an unusual shape and durable! Amazon's iphone app let you scan in an item and find it cheaper on Amazon- so instead of the higher pet shop price, I scored a savings deal on Amazon! Nice!
My big, fat, sad complaint is that you cannot CHOOSE the dinosaur! I knew this when ordering and the stegosaurus arrived- honestly I was hoping for the T-Rex or the Brontosaurus but I guess you get what you get. Still, a beagle gnawing on a T-Rex would be pretty funny...
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Reviewed in the United States on August 13, 2012
★★★★★ 5
Great Toy for Teething and Strong Chewer
Eli is now 10 months old, a labrador retriever that proves time and time again he needs toys that he can chew, but won't fall apart. He cannot have the stuffed animal toys (no, those he'll tear apart in SECONDS, never mind minutes). Aside from the Kong and the antler, this is the only toy he can chew on that won't fall apart. Even animal bones will split in minutes.
Since he was a couple of months old, he has loved this Dinosaur Chew Toy. When he was teething, I'd put it in the freezer (sometimes put peanut butter on it to keep his interest). It was not only great for teething, but also massaged his gums. Now that he's out of teething phase, he still loves to chew on it.
For the money, this is worth it. It's saved the furniture and the house from being chewed on, as he focused on this toy instead. Highly recommend, especially for the price. For any strong-chewing pup or dog in particular, this toy won't shatter, split or break apart.
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Reviewed in the United States on May 15, 2013
★★★★★ 1
Cracked Tooth
Update: While I originally loved this product and so did my dogs, I feel the need to update this review. The Dura Chew Nylabones are incredibly hard, which is what makes them last so long. However, one of our dogs did crack a tooth recently from chewing on a Dura Chew. Since then, we have been very hesitant to allow any of our dogs to chew on these as other reviews also state that dogs have cracked teeth. We have since thrown out all of the Dura Chews that we had. We are still using the regular Nylabone products (when they aren't made in China), but once those are gone we will not buy any additional products from Nylabone. We have been buying Whimzees for our dogs for the last two years or so and they are a healthy edible alternative. They do not last as long because they are edible, but I have the peace of mind in knowing that my dogs are safe with them. Another healthy alternative would be antlers, which we also give to our dogs and they usually last a long time.
Original review: Another great Nylabone product. I love that dogs can chew on Nylabone products and they can't pull off pieces like products from other brands (Hartz for example). The Dura Chews are especially great for our hard chewers and they last forever! I love all of the dimples on their chews too, they're great for keeping our dogs' teeth clean. The vet is always impressed when they check their teeth.
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Reviewed in the United States on March 21, 2015
★★★★★ 5
If Your Dog Can Rip The Hood Off Your Car--This Is The Dental Toy For Him/Her
I am always curious about buying the "number one best seller" that Amazon lists in any product category. And when it's an add-on item offered at a reasonable cost, I'm even more curious.
From reading the reviews, it appears that some dog owners ordered this item and were disappointed in it for a number of reasons, primarily because the 'hardness' of the toy caused dental problems. Obviously no one wants to buy a dental toy that actually causes teeth problems. However, some small breed owners must not be aware that their particular breed is subject to dental issues regardless of what toys they chew. Just off the top of my head, this type of problem often occurs with Yorkies, Chihauhaus, and Chinese Cresteds. You can use virtually any dental care method in existence and still watch your Chinese Crested's teeth fall out.
So small breeds shouldn't be given tough Nylabone toys like this Dinosaur model. If you watch any dog chew on this particular toy, you will quickly notice how small his/her teeth are in relation to the toy, and you will probably be surprised by the leverage and power he/she puts into each bite. They gnaw, they gnash, they continue to work at it until those tiny teeth lose the battle against the tough Nylabone. Comparing this to human teeth, everyone knows someone who fractures/cracks a tooth somehow. That's because our front "chicklet" teeth bite down on something hard, get broken from falling on your face, getting hit in the mouth, having teeth weakened by cavities, and from not seeing a dentist/hygienist on a regular basis--usually because we're afraid of a little pain.
We're always looking for relatively tough dental toys for our 182 pound Cane Corso named Dante. He tears apart any toy we buy him, usually in a matter of minutes. But this particular Nylabone Dura Chew has survived for months now. If you look at the size of his teeth, length and thickness, you will see a dog with the teeth and jaw muscles strong enough to tear the hood off your car--which is exactly what he did to our Prius. So if your dog is capable of ripping hoods off cars, this treat should be good for your pet monster. Just keep an eye on the toy so that you can toss it before it breaks down and is swallowed.
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Reviewed in the United States on March 10, 2015
★★★★★ 5
Best Nylabone Fido & I Agree
My dogs love these Dino Nylabone's. Over the years I have bought at least 30 of these nylabones for 3 different dogs. Two of which have loved the Dino shaped Nylabones the best. The third loves the nylabone daily dental bone the best but the dino is a close second. The T-Rex is their favorite but they like all dino shapes. Depending on the dog's chewing mood these have lasted anywhere from a month to 6 months before needing replacing. My super chewing husky will chew one up pretty quickly in about a month, my moderate chewing pit bull would take 3-4 months and my slow light chewing shiba inu would take 6 or more months. I typically have 2-3 chewable items per dog (it helps prevent toy hoarding and aggression) and these dino toys are always among the favorites.
*These are chewing style nylabones they are not supposed to be consumed quickly as a snack or treat. I consider these like a hobby or activity for my dogs to do not something they eat or a toy they play with. Chewing is a natural part of dog behavior and is good for dental hygiene. Giving your dog items they are supposed to chew on is a good way to prevent your dog from chewing on things he isn't supposed to.*
These are the white nylabone material which is a tougher material than the beige color nylabones. The white nylabones should be used for aggressive chewers. Beige nylabones are good for light chewers.
The T-Rex is about 6" by 5" the Long neck is about 3.5" by 7 inches and the Stegosaurus is about 6.5" by 4" they all seem to last about the same amount of time.
I can usually get them for $4-5 on amazon which makes them one of the best deals for a chewing nylabone which is another huge plus.
Overall My dogs and I love the Dino Nylabones and I will buy them as long as they are made. Great Product
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Reviewed in the United States on December 2, 2015
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