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Description
Rat ASAH2 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 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) 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 N-Acylsphingosine Amidohydrolase 2 (ASAH2) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat N-Acylsphingosine Amidohydrolase 2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Acylsphingosine deacylase 2 (ASAH2) is a protein-coding gene. Diseases associated with ASAH2 include sphingomyelinosis and lipomatosis. Pathways involved include sphingosine and sphingosine-1-phosphate metabolism and S-1P-stimulated signaling. A key homologue of this gene is ASAH2B. Acylsphingosine deacylase 2, encoded by Asah2, is highly expressed along the brush border of the small intestine, suggesting its potential involvement in the digestion of dietary sphingolipids. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.7 ★★★★★
Based on 6 reviews
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Product Reviews
★★★★★ 5
My dog loves these toys.
Size: Mini, Color: Gator (Pink)
Not a huge toy, nor is my dog. But we have lots of goDog toys. Practically indestructible and my dog likes to carry these in his mouth and bring them onto the foot of our bed. Brilliant colors and good squeaker but my dog is afraid of the squeak. Fair value for cost.
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Reviewed in the United States on June 10, 2026
★★★★★ 5
Our XL dogs love them. Highly recommend it!
Size: Mini, Color: Gator (Pink)
Our XL dogs love this! Highly recommend them! Highly recommend. Not for aggressive chewers but our big dogs don't destroy their toys. They cherish them. Great for play and cuddling. Not to loud either. Very cute.
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Reviewed in the United States on May 13, 2026
★★★★★ 5
When they said strong...
...they weren't kidding. Our pup would chew through everything she could get her sharks teeth into. Like my arms. Nothing lasted as long as a week or maybe two. But she has had Dino for months now. She loves him. He is the go to toy for when she wants to play tug. The material used to make this toy is every bit as strong as the seller claims. I will definitely buy from them again. And I will not wait for this one to rip. I may be waiting a long time. If your pup likes to tear stuff apart, feel safe in buying this toy,
UPDATE: 2/9/2023
Dino finally succumbed to the torture. His back ripped open and despite trying to repair him with ordinary thread he couldn't withstand the pressure, His stuffing was removed and spread all over the living room. Completely emptied he is still a good toy. Strong enough to withstand the forceful play of tug-o-war.
Only thing I would suggest is to use something stronger when closing his back. The rest of him is great. I will be replacing him soon.
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Reviewed in the United States on January 30, 2023
★★★★★ 4
Not so tough
Size: Mini, Color: Gator (Pink)
I bought this because I dog sit. The dog chews up every. Single. Toy. So I bought it since it was cheap and it can be in the “chunk” (the dog’s name) toy box. It is not durable at all. He tore it up just as fast as a cheaply made toy.
Chunk LOVES it! He would not stop playing with it. He loves the squeakers, it is a perfect size for him to throw it up in the air and chase it, and he loved ripping the legs off. It is good for my husband and I because there is little to no fluff.
However, it is the perfect size to drop behind the couch and not be able to get. So once he drops it behind the couch, he won’t get it until morning.
Now for my dog’s rating. My dog loves this toy too. He is much more gentle with toys (he has $5 Walmart toys we bought him back in 2020 in perfect condition that he plays with them every day). Before Chunk came over for the weekend, my dog Ted played with this. Ted is a larger small dog and chunk is a smaller small dog. And it is still the perfect size for both dogs.
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Reviewed in the United States on April 26, 2026
★★★★★ 5
Tough
Size: Large, Color: Dinos Frills (Pink)
Tough dog toy that's held up through multiple washes! My dogs love the squeaker. They are not destructive chewers so I can't really say how it would hold up with that kind of dog though.
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Reviewed in the United States on June 8, 2026
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