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Description
Human ABHD5 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 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 capture antibody against abhydrolase domain-containing protein 5 (ABHD5). 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 abhydrolase domain-containing protein 5 (ABHD5) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Abhydrolase domain-containing protein 5 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | αβ-hydrolase domain-containing protein 5 (ABHD5) is an enzyme encoded by the ABHD5 gene. The protein encoded by this gene belongs to a large protein family defined by an α/β-hydrolase fold and contains three sequence motifs corresponding to the catalytic triad within the esterase/lipase/thioesterase subfamily. Unlike other members of this subfamily, its putative catalytic triad contains an asparagine rather than a serine residue. Mutations in this gene are associated with Chanarin-Dorfman syndrome, a glyceride storage disorder characterized by impaired long-chain fatty acid oxidation. | |||||||||||||||||||||||||||||||||
| 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 1772 reviews
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Product Reviews
★★★★★ 4
Not real Black Iron, but works just as well
Size: 42inch Tall (NOT included wood planks), Color: Black
First, these parts that I got were not real Black Iron pipes like you would pick up at a hardware store, they are like furniture grade tubes for an IKEA shelving system. The thread would fit 3/4 pipe from like HD, but they are not the same, I know this because I just bought some of the pipe and parts for a small shelf right before ordering this. I should have known from the price, the real stuff is kind of pricey, but nicer. The elbows and T-connectors as well as the flanges seem more like galvanized steel painted black. HOWEVER, the end product is pretty nice and very versatile as to where you would want to put it. It is still pretty sturdy, and if screwed into a stud, I believe it would hold a lot of weight. The entire shelving system is painted black, so unlike Black Iron, it will not stain your hands black when handling it. Putting this together took about 30 min, the longest part was adjusting angles and lengths to get it flat and squared, but was not hard at all. This kit does NOT come with shelf boards, so length of that is up to you. It also came with mounting screws, but they are thin and I would suggest as your getting boards for shelves, also pick up some beefier screws, U used #12 - 1 1/4" wood screws with the sleeves for drywall and a little longer for securing it into a stud. All in all, this black pipe shelf looks nice, is nice and will work out great, just know, it is not the real Black Iron pipes and parts you would get from a hardware store, but the threads would fit if you wanted to add to this. I only knocked down one star because of this, and if I am going to buy more of these, I would prefer the real stuff and not go with this one.
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Reviewed in the United States on June 5, 2020
★★★★★ 5
Love these!!!
Size: 42inch Tall (NOT included wood planks), Color: Black
Super sturdy..looks excellent. Industrial and clean lines....lots of space for our kids bathroom. The wood does not come with it but was easy to get lowes. We actually added another shelf on the top bracket to make 4 shelves. They're nice and wide to fit baskets and organize things. For teenagers bathroom! My husband installed by himself and he isn't the handiest of guys but it it wasn't bad. I do recommend getting pipe fastners to secure the boards to the pipes so they don't shift around
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Reviewed in the United States on June 2, 2021
★★★★★ 5
Nice pipe shelves! Excellent quality!
Size: 28inch Tall (NOT included wood planks), Color: Black
I really like the fact that the pieces were already painted. I compared the cost of the unit to buying the pieces from the big box store and this was far more cost effective for me.
The installation went well and I ended up creating a template for installation. For the template, I measured the width of one of the flanges (part that mounts to the wall), cut a piece of material the same width. I found the center and drew a line down the middle. I then placed the section with top and bottom holes over the center line and then marked the other two holes.
I then drilled out the holes, put on wall and used level to plumb. I used small pilot drill bit to drill through the template into the wall.
One thing to note is that I did not use the included anchors instead opting for some from the big box store.
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Reviewed in the United States on March 29, 2020
★★★★★ 5
Well built and priced
Size: 42inch Tall (NOT included wood planks), Color: Black
I saw these on line and decided I could probably just build one on my own. After pricing the parts out at the local hardware store, it was almost the same price, and opted for the less hassle pre-fab online kit. The material is well made, same as the hardware store, except the threads are shorter for easier assembly, and its painted for a cleaner look. I bought the 12"Dx42"L kit, and although I knew the measurements, it was a lot more massive than it looked in the picture. An 8' or 10"x 24"L kit would have fit my needs better. I only assembled 2 of the 3 sections, and will use the single section leftover for another shelf elsewhere, so it wasn't a problem. I'm happy with the end result and would recommend.
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Reviewed in the United States on June 28, 2020
★★★★★ 5
So beautiful!
Size: 42inch Tall (NOT included wood planks), Color: Black, Size: 42inch Tall (NOT included wood planks), Color: Black
The poles are very sturdy. The order doesn’t come with wood but I only spent ~$15-20 on the wood and stain needed to shelve the poles. The only complaint I have is it was hard to install; mostly due to my lack of experience. It took most of the day to install and stain and cute the shelves. The poles weren’t completely flush to the wall and I couldn’t figure it out for the life of me. I love the way these turned out and I know these shelves will be coming with me to the next home I have. 10/10 would recommend, even with the hard installation. SO worth it.
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Reviewed in the United States on July 26, 2019
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