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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 344 reviews
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Product Reviews
★★★★★ 5
5 stars
Format: Kindle
Great conclusion to the series.. with an all-star cast involving the extended Bat family.. reminiscent of the "Battle for the Cowl" and "Return of Bruce Wayne" days in scope and back story.. definitely a must-read!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 16, 2017
★★★★★ 5
Great story by several great storytellers
Format: Paperback
Picks up where vol1 left off but generally feels more cohesive and organized. Great story by several great storytellers. Connects heavily with Grayson if you want more context.
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Reviewed in the United States on February 27, 2017
★★★★★ 3
another chapter in the Batman story
Format: Kindle
Was good but I didn’t love it. There were definitely some high points but I just was not glue reading the next part every time. Some of it was the art teams were also highs and lows. When the art was better I did find myself more engaged with the story. Also to be fair when I read different volumes I have to at time get caught up on which universe version is this going on from. Sometime it can get confusing if your an older read like myself and you have tons of other canon that does always fit in.
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Reviewed in the United States on September 2, 2025
★★★★★ 5
An Epic tale and more...
Format: Paperback
I couldn't be happier with this book. Not only does this carry the cosmic Spidey issues that crossed over through the three Spidey books of the time, Amazing, Spectacular, and Web of Spider-Man, but also the 1990 annuals of each book, which had our hero shrunken down to the size of an insect and smaller, fighting alongside Ant-Man against would be technology thieves and then through the Microverse. We have the full annuals so there's even stories featuring Mary Jane, Aunt May, and others in the Spidey universe.
With the inclusion of the Punisher and Venom Amazing Spider-Man issues, it almost feels like three trades in one thick book of Spidey goodness. The art is fantastic also. From Sal Buscema's underrated Spectacular series, to Erik Larsen's Amazing series, and even Todd McFarlane's last Amazing Spider-Man issue where Spidey punches The Hulk so hard, he leaves him orbiting Earth!
While this book has several writers and various other artists, I still find this to be a cohesive collection well worth the price of admission.
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Reviewed in the United States on December 12, 2013
★★★★★ 4
Spidey SMASHES Hulk
Format: Paperback
This book presents nearly 500 pages of Spidey Comics from 1989-90, Collecting Amazing Spider-man 326-333 and Annual #24, Spectacular Spider-man 158-160 and Annual #10, and Web of Spider-man 59-61 and Annual #6.
The big event of this comic ties into the much larger Acts of Vengeance story arc. Several supervillains team together, realizing that they've been losing to the same people for 25-30 years. They come up with the idea of trading and going after each other's enemies, thinking that the heroes will not know how to react. (Apparently, it never occurs to them that they will also not really be able to respond to the heroes techniques.) Because Spidey at that point had three magazines a month, that met he'd be hit with three times the rivals.
But after serving the first attack of Graviton, Spidey has an accident that ramps up his powers and makes all attacks on him go very badly for the villain with one villain even getting accidentally killed in the process.
I have to admit that there was something wonderfully pleasing about Spidey knocking around the likes of Magneto and the Hulk like they were rag dolls. Seriously, the first nine issues in this book, are Spidey kicking one threat after another as he has power on par with the Silver Surfer.
We don't learn until the last issue the real reason for the power and longtime readers had to be scared that this was another alien costume, and in a way it was, but if this was like the symbiote, it was a good force that bestowed the uni-power when it was needed. The whole thing has a pretty satisfying ending.
Probably my chief complaint with this book is that the true core of the Cosmic Power ends on page 210, really, AS #329-333 have nothing to do with the Cosmic Powers story and the Annuals are very vaguely related.
AS #329 and 330 is a somewhat violent (but not overly so by today's standards) crossover with the Punisher battling drug cartels and a US government plot to smuggle drugs. The story has some serious moments but ends with one of the goofiest concepts in comics ("Cocaine Standard" 'nuff said). Issues #331-333 is solid story of Eddie Brock/Venom escaping prison and it's interesting in its own right.
There's a three part story spread across all three annuals in which Spider-man is shrinking. At first in the Amazing Spider-man Annual, it looks like it's because of inhaling Ant Man's shrinking gas but it's not that at all as we find out in the other two annuals. The story is decent enough, though Marvel's decision to make people buy all three annuals back in 1990 was somewhat chintzy, though defensible since the story runs 70 pages. For 70 pages, it was good but not great.
However, Marvel actually reprinted everything in the annuals which is a bit of a mixed bag for readers. On one hand, you get the full Annuals with all the extras. On the other, it breaks up the "Spidey's Totally Tiny Adventure Story" and you get a very mixed bag of extras. My thoughts:
"The Mercy Bomb"-A story told in part by Spider-man co-creator Steve Ditko. Seemed to have an anti-war message but didn't tie into anything and was just blah. Grade: D
"A Time to Choose/The Choice":Whatever can be said for spreading the 70 page Spider-man story across three annuals. There was really no reason to break this story of a reformed Sandman facing a tough choice when he's offered a chance to go back to the old life of crime by the Trapster and the Wizard. It's an okay story but seems a little forced. Grade: C+
"Pete and MJ's New Pad"-After the loss of their condo to an unethical real estate developer, Pete and MJ moved into a new apartment. This special feature took a look at the apartment revealing that it's an average apartment with nothing interesting in it. Grade: D
"Amazing Fantasy"- A not so Amazing dream sequence filler. Grade: F
"Pale Reflection"- Former Spider-man villain Hobie Brown goes on a job and learns that he can get beat up. Really? Grade: D-
"What I Did on My Summer Vacation"-A ten page story featuring juvenile delinquent turned crimefighter Rocket Racer, who'd appear in Spider-man: TAS. An okay but not great story. Grade: B
"Sales Day for a Shootout:" Aunt May helps the Punisher kill terrorists. Actually surprised at how positively the Punisher was portrayed in this. Grade: B+
"Eleven Angry Men and One Angry Woman:" This take on Twelve Angry men as Mary Jane as the only hold out on a jury ready to acquit a defendant who claims Spider-man was a thief. There's some humor and a little bit of poignancy. Of course, the wife of Spider-man shouldn't be on this jury but it was a fun story. Grade: A-
"Child Star"-One of the weirder stories in here involves a two year old getting the unipower that Spidey had because a couple summoned demons to help play the stock market. Grade: D
The book ends with material from the first Trade Paperback printing of the main 9 issue Cosmic story which means that you get to find out the background of the book after it's over.
In addition, there's a lot of ongoing plots in this book that were dropped into the middle of because of comic continuity. Joe Robinson is in jail and we really don't know why. Aunt May's friend Nathan is dying but we don't know when she met him or how deep their friendship is. The Black Cat begins to get, well catty, about Peter having married Mary Jane and threatens to break Flash Thompson's (now Peter's best friend) heart out of spite. However, this is just the nature of jumping into an ongoing comic book story.
That said, with all the things I've mentioned, I can't bring myself to rate this less than 4 stars. The core material is awesome and so are most of the actual Spidey stories outside of it, despite the uneven nature of the non-Spidey stories. What's particularly pleasing is seeing the Parker-Watson marriage for fans of that relationship that was abandoned with One More Day. It's not a perfect marriage, but it's clear that it's a positive in Peter's life and it's written way that's not glamorized but is appealing.
If you can take the book's hiccups, this is a good book for teenagers and adults.
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Reviewed in the United States on April 21, 2014
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