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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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4.8 ★★★★★
Based on 40 reviews
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Product Reviews
★★★★★ 4
A Decent Fae Romance
Format: Kindle
** 3.5 Stars rounded up to 4 **
First off I want to say that I found the author, J. D. Linton, on TikTok right before the release of the sequel. I bought the first 2 books to support her release, so congrats to her for the release of the sequel!
Overall, I liked the story & plot, I liked the characters, and I liked the spice. The downfalls: not enough development between the characters for the romance, and it leaned a little heavy into the tropes. A lot of stuff is told to you, which could have been used as devices to drive the plot and emotions of the characters.
🛑🛑 Mild Spoilers ahead, I tried to hide the obvious ones 🛑🛑
This is very obviously a fated mates, which is a trope I usually really enjoy. But I wish the relationship between Ara & Rogue had much more development before it's realized that they're mates, instead of Rogue knowing almost immediately and using it for nefarious purposes (at least in the beginning before they get to know each other). By revealing this within the first quarter of the book, I feel like it leaves less room for them to fall for each other organically (albeit with help from the mating bond) and they love each other because of the mating bond. I was disappointed as soon as Rogue know (literally only 5% in) and I literally made a note: "As much as II love a good mating trope, I wish we had to work for it a little more. Where's the fun in just telling us?" I believe that by holding out and feeding the reader snippets of a potential bond, it would've been more rewarding as a reader.
I also wish there was more world building - we are told of a war between human & fae but don't get to really experience any of it. Ara is sheltered in her human home, then sheltered in Rogue's castle. There's bits and pieces about what the war has done on either side - but we're more told of the aftermath and don't really experience any of it. Ara's father is supposed to be the king's #1 general - yet he is at home with his family & with Ara for the first couple chapters. Her, her family, nor her village seem to be affected by the 10 year war going on on their borders. I wish there was a little more setup to make this conflict - an actual war - feel more than a skirmish between fighting territories.
Linton could also be a little repetitive - with the biggest culprit being when Ara is upset she "brings [her] knees to [her] chest]" and either sits like that or cries. Every time she is upset this phrase is mentioned. I would get it if this was her crutch, or how she copes with grief and stress, but that should be explained why she does it so often or it becomes repetitive. I started to get annoyed with how often she would sit like this solely because it happens every couple chapters.
However, I did really like the spice. I love an enemies to lovers trope, especially when it results in spicy scenes. The spicy scenes weren't anything new, but they were fun. Wish there were more but that could also just be me - there is no such thing as too much spice 😂
Would I read again? Probably not, I'm super picky with rereads. Although I did genuinely enjoy my first read through!
Will I continue the series? Probably, at least for the sequel. As for #3, kind of depends on where I am with my TBR once that is released
All in all an enjoyable, fast paced read
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Reviewed in the United States on March 8, 2024
★★★★★ 3
I love indie authors
Format: Kindle
Let me preface this by saying—writing a book is HARD! Coming up with characters so real that they take on a life of their own, building an entire world, the political/magic system. Designing all of this is no easy feat.
That being said.
This book had so. Much. Potential. I was so excited to read this book, and I plan to finish it for the sake of finishing it. But. At this point, I would have set I’d aside as a DNF. The book could have benefited from some form of a developmental editor, or an in depth beta reader. I will say this. Within 5 chapters, there are so many… phrases that I’ve highlight that I’ve latched onto. Phrases about books and storms that were written BEAUTIFULLY! So, bravo Linton for hitting the nail on the head as to why readers disappear like they do!
However, 5 chapters in and I can already guess where a majority of the story is going. But that’s also because I read like a mad-woman and have read this particular type of story, many different ways. Enemies to lovers where the FMC isn’t who she thinks she is. I am all about supporting indie authors. BUT. I also feel like criticism should be constructive, and not degrading. So if I could give this book a 4 1/2, strictly because I know the work the author put into this, I would. So if you’re looking for an easy read with characters that aren’t hard to follow, look no further! They are easy to love and easy to care for. One of the biggest issues they lacked, to me, was depth and plausible reactions to their situations. JD, you have done BEAUTIFULLY writing this book. I applaud and will continue to buy your books in the future. My BIGGEST recommendation is to definitely hire some form of an editor for any upcoming books. Or in turn, I will be happy to beta read for you. Should my opinion change of the book by the time I finish, I will happily get on here and say I was wrong, delete this review and post a different one. Until then…
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Reviewed in the United States on November 20, 2022
★★★★★ 5
this book set my soul ablaze! <3
Format: Paperback, Format: Paperback
"i had never really cared about the weather before, but now, clear skies meant everything to me, and i was grateful to see another calm morning."
this book. this book!
i loved the last storm so much. the writing style. the descriptions. the world-building. the characters. the plot twists. the tropes. the sexual tension. the—everything. everything was magic.
the last storm follows our two main characters, ara and rogue, giving us dual POV from both characters (which i loved, btw). ara, a human girl who has been locked away in her father’s estate most of her life, just wants to see the world. all she dreams of is seeing what else is out there. but when her father announces her engagement, she knows that dream will become nothing more than just that—a dream.
rogue, the fae king, is tired of the attacks being rained down on his people. in hopes of finding out the human king adon’s secrets, rogue infiltrates auryna’s borders. in his last resort to gain information, he visits the local pub. to his surprise, the general’s precious only child is sitting at the bar, drink round after round of mead. now he just needs to figure out how to take her without anyone noticing.
first and foremost, let’s talk about the endless list of my favorite tropes and aspects that this book had.
›› enemies to lovers
›› fated mates
›› one bed
›› the chosen one
›› elemental magic
›› actually good and shocking plot twists!!!
›› badass female lead
›› morally-grey love interest
›› fae/human war
›› force proximity
›› touch her and die
›› who did this to you?
›› captor/captive
›› praise k!nk (panting profusely)
“you are entirely the opposite of everything that i am, and i would gladly wear your shackles if it meant i could have you.”
it’s been a long while since i read a book i liked this much. but i just loved this book. it set my soul ablaze. thank you to the author for writing this beautiful story and for blessing me with an eARC! i loved it so much that i immediately bought the paperback upon release!
every aspect of this book was just beautiful. i was blown away by the way the world was described, the way feelings were portrayed, the way the elements were used in the fae’s magic. it just—AHHH! i just absolutely adored it all. i cannot wait for the second book to release next year!
also the way he calls her “little storm” sets my heart on fire.
this was a fast-paced read and if you are a lover of acotar, fbaa, deal with the elf king, or any other similar books, then please stop everything you’re doing and read this book right now. you won’t regret it.
thank you again, jd linton, for giving me the privilege of reading your arc and for blessing this world with the world you created. <3
"something about him pulled me in, like a moth to a flame, and it felt as if i was just waiting for the inevitable burn that came with flying too close to the fire."
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Reviewed in the United States on November 15, 2022
★★★★★ 4
Fun, both heartwarming and heartbreaking
Format: Kindle
Only my second first person written selection, I am still getting used to that aspect, but unlike my first, I enjoyed that the story was told through both MCs. A great enemies to lovers, forced proximity, fated love etc, that resonated to me. There were some small twists that I could see coming, but also a few that I didn’t quite see until the characters were also seeing. Personally, I am more interested in the story than the spice, but with that said, it was well seasoned! I am kind of new to the spice world so I can’t say for sure how this would rate, but it definitely had some heat. I am very glad I happened across this author, and I do plan on also reading the next book….if nothing else, just to see for myself the “transformation” of the characters I’ve grown to love!
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Reviewed in the United States on May 23, 2024
★★★★★ 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!
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Reviewed in the United States on December 5, 2022
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