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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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4.1 ★★★★★
Based on 2447 reviews
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Product Reviews
★★★★★ 5
😍😍 BEST DEBUT NOVEL EVER
Format: Paperback
I’m having trouble finding accurate words to describe the way this book made me feel, but I am going to do my best.
To start off with basic elements, the character and world building are phenomenal. I feel a strong bond to not only the two main characters, Ara and Rogue, but to each and every character introduced throughout the book. The author did a stellar job of giving each of them unique personhood. All of the scenes are beautifully described. So much so that throughout the entirety of the book, I could see every scene: the towns, the castles, the meadows, the landscape. I have had difficulty with this and with distinguishing between outlying characters while reading in the past, but I did not have to think to remember details of world or character building because they flowed naturally within the story and were described well. I have read book series before that made me want to be a part of that world, but I actually felt like I got to step into Auryna and Ravaryn!
The plot twists!! Although this is not a suspense novel, it still had me on a rollercoaster of emotions and on the edge of my seat from beginning to end. I haven’t cried actual tears over a book since I was in high school (and I’ve read a LOT). This book finally broke the floodgates in the final few chapters. Multiple times. And we love a good cliffhanger. It truly made me FEEL.
THE SPICE is a solid 3.5/5. Some of the scenes had me flushed, some had me taking notes, some just had my jaw slack and my mouth hanging open. Bravo, JD Linton, bravo.
The relationships: friendships, family, romantic, ALL of the relationships in this book have so much meaning. The author does a great job at making you feel the love, the anger, the peace, the frustrations, the safety, the familiarity, etc. between the characters.
Ara and Rogue. I can not say enough and I also do not want to say too much. Just know that I feel like I know them both, to their core. I know what their childhood looks likes, their darkest moments, their biggest fears, their dreams and passions, what they want in life… The POV switches were seamless. I am so happy this author decided to let us see from both sets of eyes.
I can not wait for book two after that cliffhanger. And there is SO much potential for at least one prequel, I can’t wait to see where this author goes! I hope this series continues and flourishes. Fingers crossed!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 5, 2022
★★★★★ 5
Amazing!
Format: Kindle, Format: Kindle
This book was phenomenal, I devoured it within a few days! For this being a debut novel, it is fantastic and I would’ve thought the author was a seasoned author. I have zero complaints about this book.
Let me start by saying that the world building was phenomenal. I could picture everything in my head because of how detailed it was — that’s how good it was written. And I absolutely love the “captive/captor” trope so much, it’s become one of my favorite tropes, so I was pleasantly surprised to see that this book had that.
I loved the banter between Rogue and Ara — they’re both snarky and witty, plus with the romantic tension, it made the dialogue that much better. Speaking of romantic tension, yes there is spice but not so much of it that it overrides the plot, which I loved. For me, this would probably be on the 3/5 level of spice.
This book had a ton of plot twists and I thoroughly enjoyed it.
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Reviewed in the United States on November 13, 2024
★★★★★ 4
High on Tropes and Satisfaction
Format: Kindle
This is a great Romantasy book full of action, adventure, and everything you look for in this genre.
I won’t lie: it does kinda feel like the author found every common trope from every successful book of this kind and threw them all into this novel. But if it ain’t broke, don’t fix it. Especially in romance, there’s a large audience who has specific expectations, and they want them every time. Nothing wrong with that and many times I’m one of them.
I have no idea what defines a spoiler honestly, so
spoiler alert!!!!!!!
Tropes include:
Only one bed at the inn/bar
Dissatisfaction with life before hunk appears
Lost royalty
The chosen one
Montage of dress up time followed by shocked hunk
Forbidden romance between two from rival peoples
Power that cannot be controlled, simply guided/asked
Gathering intel at the inn/bar
FMC who knows how to fight/use weapons well
There’s probably more but no need to list them all.
Good story and I would recommend!
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Reviewed in the United States on June 14, 2024
★★★★★ 5
Great start
Format: Kindle
I loved this book! It's funny but still deals with tough themes, like chronic illness, a serial killer on the loose, and a dash of self-harm. The guys are interesting and distinct, we don't know too much about them yet. It does end on a really terrible cliffhanger but on the bright side the next book is out and I believe the series is complete. I have enjoyed both of these authors separately and this is a great team up!
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Reviewed in the United States on May 2, 2025
★★★★★ 4
Fudge!
Format: Kindle
Titles aren’t my strong suit. Sorry not sorry.
That ending has me completely frustrated book two isn’t out yet. Just throwing that out there.
First thing first, this is a high school book where the lead is under 18. Yea yea she’s about month from being an adult, but I can’t say I would’ve even opened the file if I’d realized. It’s not a smut fest or anything, but there is sexual tension as each party figures out their emotions.
That being said, it’s a good book. There are some troubling parts, like how the supes come into their powers at 13, but most of those are dealt with in a way that makes sense. Kian is the exception. His whole arc pisses me off, especially since no one stepped in. Reflecting back on real world situations, ten or so years ago, I can see it happening, but it still makes me sick. Which, I’m sure was the whole damn point.
For the most part, Courting Darkness is a fun read. I found myself laughing along with most of the set up. By the time it started getting serious, I’d grown to like the characters enough it held an impact.
I’ll be adding book two to my list for when it comes out.
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Reviewed in the United States on March 8, 2022