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Description
Human Cyclin D1 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 freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes 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, but avoid repeated freeze-thaw cycles. Pre-Assay Preparation: 1. Remove the reagent 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 stand for 15 minutes to completely dissolve, then gently mix (concentration 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 seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 20ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 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 cyclin D1 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 cyclin D1 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cyclin D1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Cyclin D1 is a protein encoded by the CCND1 gene. The CCND1 gene encodes the cyclin D1 protein. This gene is located on the long arm of chromosome 11 (band 11q13). It is 13,388 base pairs long and translates to 295 amino acids. Cyclin D1 is expressed in all adult tissues, except cells derived from bone marrow stem cells (both lymphoid and myeloid lineages). Its overexpression has been shown to be associated with early cancer onset and tumor progression, and it can contribute to tumorigenesis by increasing anchorage-dependent growth and angiogenesis through VEGF production. | |||||||||||||||||||||||||||||||||
| 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 homogenate, cell culture supernatant |
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4.5 ★★★★★
Based on 1524 reviews
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Product Reviews
★★★★★ 2
Not that great
Huge "The Simpsons" fan. I watched all the episodes and read a lot of the comics and enjoy most. This one however was quite boring. Turns out I'm just not a big fan of Chief Wiggum. I really enjoyed The "Homer" and "Bart" book. But not this one. Maybe if you love Wiggum you'll love it. If you're not too fond of him then I wouldn't buy it.
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Reviewed in the United States on March 28, 2013
★★★★★ 3
Good for my collection.
Format: Hardcover
This was another quick and entertaining read. This instalment wasn't as enjoyable as some of the other books but it was still good. I'm glad to have it in my collection.
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Reviewed in the United States on May 27, 2014
★★★★★ 5
Alan Moore Does it Right, and Bolland Art Revision is Amazing
Format: Hardcover
The Killing Joke was a comic spawned shortly after the work of Frank Miller, and is probably the comic that established The Joker as one of the most important fictional villains of the 20th century. Batman had become a well thought out, complex character in recent years, primarily due to the work of skilled writer Frank Miller. Batman had been brought into a gritty, modern world of comic books, but I always felt that Miller's The Dark Knight Returns failed to bring the antagonistic Joker into a new light...well it did, but not in the way the character was meant to be. Alan Moore, with the help of Brian Bolland's stunning artwork helped bring to life what is widely accepted as today's standard for the famous Harlequin of Hate.
He's crazy, he kills people simply for his own twisted sense of humor, but always has a very zany, cartoonish attitude, which I felt was lacking in Miller's interpretation, where Joker seemed much too serious in appearance, dialogue, and action. The Joker presented in this graphic novel truly is a fiendish jester of fate, who for the most part is truly frightening, but never really acts like anything more than a loon. Bolland's art is very helpful in this aspect, not only bringing the Joker's exaggerated, skeletal body to life, but making it perhaps the most anatomically correct Joker ever seen, while still following the style of the 1970s Joker appearances.
The story revolves around the long struggle between Batman and his foe, and their never ending conflict. Joker, having escaped from Arkham Asylum (again), has decided to prove that one bad day can transform any sane man into a monster, such as himself. Taking possession of an old carnival he's ready to do whatever it takes to prove his point, with the assistance of a circus freak show. Meanwhile The Dark Knight questions himself on how this fight will end; coming to the realization that sooner or later one of them would kill the other, unless they tried to reason it out. A hopeless plan by our hero, but he has to at least try a single time to reason with his arch-nemesis, just so he can say he tried.
While Batman leads the hunt for the psychotic clown, Joker decides to prove his theory, kidnapping Commissioner Gordon, and shooting Barbara Gordon (Batgirl) through the spine (she never walks again). This is one of the most villainous acts in the history of comics; simply because of how pointless the shooting was, considering Barbara wasn't even in the character of Batgirl. She was just a bystander who Joker decided to use as a tool for his plot. Everyone is a puppet, or tool in his mind.
He does this act all of this while wearing a stereotypical beach-going tourist's attire, complete with a large camera, the only exception being his wide-brimmed, purple hat, which conceal his eyes. These are the eyes of a madman, and in one of the comic's final pages his eyes are concealed in shadow once again, before they come out and you see him for the hopeless case he truly is.
The story sets the stage for the most widely accepted Joker back-story through a series of flashbacks, showing him as a failed comedian with a pregnant wife, and soon winds up assisting criminals, and falling victim to tragedy. The character is shown to have had one bad day and that is how he ended up as the smiling super villain who has plagued Gotham City for decades. Of course this back-story isn't necessarily true, being that it comes from flashbacks spawned from Joker's mind, and as he famously states in his confrontation with Batman "If I'm going to have a past, I prefer it to be multiple choice!"
Gordon's capture and torture leads to a fight between these two iconic characters, where Batman tries to see if it is possible to reason with Joker logically, but the villain doesn't even try to lie about what is obviously in store for them. He can never be reasoned with, and won't stop until he's dead, and Batman refuses to kill him so it's a sick cycle that goes round and round. In the end of The Killing Joke the roller coaster has only been reset and it will only be a matter of time before Joker is back on the streets, killing again with some new, random scheme which will only make sense to him alone, depending on the mood he's in at the time, or which version of his past he remembers.
Alan Moore's story is sick, it's disturbing, and doesn't fail to leave the reader in a state of awe. The only problem is that by the end you crave for more! The story practically brings you to the point of begging for a sequel, or expansion which will probably never come. This is a one-shot story, and should remain that way, or else the original's impact may be lost. I don't have a single friend who doesn't think The Killing Joke is one of the most twisted, sick, most perfect stories of the comic medium. It's short, but it delivers a powerful punch!
Brian Bolland's art makes this short graphic novel a masterpiece, providing some of the best art ever seen in the characters' long history, only rivaled (in my opinion) by the art of Alex Ross. Joker really is frightening to look at here, because he doesn't look like a real person in most aspects, but Bolland manages to make this character incredibly believable in appearance, without taking any liberties of changing the body type into something more universal. The tall, skeletal body of the 1970s comics is preserved for the most part, except for the narrowing of the chin, which improves the look even further (hence why it's universally used in today's comics).
Recently I got the hardcover anniversary edition of The Killing Joke, with the artwork being edited by Brian Bolland, and re-colored by Bolland personally the way he intended it to be seen. I must say that I love the changes to the colors, more so than the cheery, bright colors of the original, which I felt distracted from Bolland's line-art, because the colors were way too traditional comic book, not fitting the intensity of the art. These colors are dark, gritty, and really captures to mood of the story more effectively than the bright colors of the original release. I especially love the flashbacks being in black and white, except for concentrating on objects that reflect the color red, which leads up to him taking on the garb of The Red Hood, before his first confrontation, leading to his ultimate disfigurement and insanity. This is a seriously talented artist and I only wish he was presented with more opportunities to draw these characters. Batman and Joker look their very best in these images, and I'd be hard pressed to find a comic with better consistency of art quality.
The Killing Joke is one, if not THE best story in the history of Batman comics, and rightfully earns my rating of 10/10 stars.
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Reviewed in the United States on November 11, 2008
★★★★★ 5
One REALLY bad day.
Format: Hardcover
"The Killing Joke" is widely considered to be the be-all-end-all of Joker stories, so what better way to pay homage to the greatest comic book villain of all time near the eve of his re-unveiling in
than by reviewing his definitive story? This is the comic that (sort of) revealed the origin of The Clown Prince of Crime, humanizing him to an extent never before, and truly examined -with pictures rather than words- the antagonistic symbiosis that exists between Joker and his arch nemesis, The Batman. A beloved Gotham regular will never be the same and another will be put through hell before this story is done. Oh, and there are creepy little henchmidgets as well. Gotta love the henchmidgets.
The art is outstanding, the storytelling superb, and the character examinations are vital to understanding both combatants. The "one bad day" premise highlights the "two sides of the same coin" argument that Batman and Joker are in fact more alike than dissimilar. As if Bruce Wayne took a right when his arch-nemesis took a left. The controversial ending leaves little doubt as to Alan Moore's take on the debate, and I like it like that. While many critics have strongly resisted both the comparison and the somewhat sympathetic look at The Joker's past, the truth is that every great character -villain or hero- needs that sort of intricacy to their story to remain relevant in the world of modern fiction. Comics are no longer for children and adults realize that the world is seldom black and white, that all monsters were once men, and that unspeakable darkness and insanity resides deep inside each human mind. It can take years of suffering to bring them out or it can take one bad day. One bad day could ruin your very existence and everything you were; it's a frightening reality that cannot be overlooked while reading this comic. The more the reader is willing to ponder the ideas put forth by this story, the more you are likely to appreciate "The Killing Joke". An outstanding achievement in storytelling any way you look at it.
I was tempted to knock this down to four stars because with this book you are buying a single issue of a comic for what you could easily pay for a full trade paperback or graphic novel of equal quality like, say, Frank Miller's
, which is so good it may very well cure cancer (can you prove it doesn't?). But the fact is no Batman fan should be without "The Killing Joke" and I would rather stick to reviewing based on quality rather than haggling over price. The bonus story at the end (written and illustrated by TKJ artist Brian Bolland) is a killer little mini-comic that serves as a perfect companion piece to the main story and definitely sweetens the pot for those wondering if they should get this hardcover edition. "The Killing Joke" is an absolute mindless must-have for fans of the comic book medium and even more so if you claim to be a follower of The Caped Crusader or his twisted nemesis. End of story.
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Reviewed in the United States on July 14, 2008
★★★★★ 5
Alan Moore does it again
Format: Hardcover
Batman: The Killing Joke is by far one of the greatest one-shot graphic novels ever written, Moore is fortunate that it was of such caliber as to deserve inclusion in the mainstream Batman canon.
The Joker is the single most evil non-superpowered being in the DC universe, and almost nothing is known about who he was before becoming the arch-nemesis of Batman. While curiosity abounds for fans, even more prefer that he maintain this aura of mystery since it is believed that his pre-villain life was not one that would have been considered anything worth writing about; it is because of he IS the Joker that he means anything in the DCU at all.
All that aside, Alan Moore - the creative genius behind such works as
,
,
,
,
and many others - took a chance and in 1988 presented the comic community with what DC has accepted as the official Joker origin story.
WARNING: SPOILERS AHEAD
The Killing Joke is about madness. The Joker has escaped from Arkham Asylum and is setting about a new and truly evil scheme; unrivaled since he murdered Jason Todd/Robin #2 in
. While Batman is frantically trying to track him down, the Joker has purchased a dilapidated carnival (like something you'd expect to find in a 21st century teen slasher flick, but in his hands is decidedly worse) and is now off to secure his "main attraction". Later at the home of Commissioner Gordon, Barbara answers the door to find a sick fixed smile shooting her through her spine, crippling her; after several off-color disabled jokes, Gordon is kidnapped and spirited away to the the Joker's carnival of horrors. (Moore and DC received a hailstorm of praise and criticism for rendering Batgirl a parapellegic, but the decision stuck and arguably many good, if not great, stories came from it.) Gordon regains consciousness to find himself being stripped down by a host of sideshow freaks and lead at the Joker's behest to a House of Horrors ride filled with the Joker's own snap-shots of Barbara fully undressed and in pain and filled with the Joker and his cronies singing a sardonic song about lunacy (GOD I WISH I KNEW THE TUNE!!!); all of this intended to drive Gordon insane.
Batman uncovers the Joker's plot and rushes to the carnival to stop him; a chase ensues through a booby-trap-laden house of mirrors in which the Joker states that he's proven his point with Gordon's unquestionable descent into madness: "All it takes is one bad day to reduce the sanest man alive to lunacy!" The joker goes on in his analysis with Batman, stating that he too is crazy, but won't admit it and tries to get him to accept it. The final confrontation between them is left a mystery, but it is clear that the Joker - after decades of campy portrayals - has most definitely re-earned the title of most evil comic book villain.
Throughout the story, the Joker offers flashbacks to his life before becoming the monster he is today. He was once a technician at the ACE Chemical Plant who quit his job to become a stand-up comedian. Unfortunately he fails miserably and worries about how he's going to support his very pregnant-in-the-late-trimester wife and soon-to-arrive baby. He decides to make fast, easy money by throwing in his lot with criminals looking to rip off a playing card company next door to the chemical plant and he offers his services to get through unnoticed. So as to protect his own identity, the gangsters present him with his disguise: The Red Hood. While in discussion for the crime, a couple of cops show up to tell him that his wife has died while operating a defunct bottle heater. In his shock, he tries to back out of the crime since he no longer has a reason to go through with it; but the gangsters hold him to it. Later on, they break into the plant, but everything goes wrong as the cops show up and take down the gangsters and that Batman appears to apprehend who he believes to be the Red Hood. Whether he jumped, fell or was pushed is unclear, but the man landed in a vat of chemicals that washed him out of the factory. When he reaches shore and removes his disguise and sees his reflection in a puddle of rainwater, he begins to laugh, turns and reveals himself as the Joker.
At the end of this story follows another one from the mini-series
called here "An Innocent Guy" about a man - clearly disturbed, making the judgment that if anyone is to truly live a life devoted to good, then they must commit an act of evil to know that that is what they really want. After laying out his basic concept, he arrives at the conclusion that he must kill Batman. He lays out his plan in disturbing detail and closes with the a that after this one act of evil, he can go on to live a thoroughly good life and go to heaven when he dies.
Masterfully written by Alan Moore and beautifully illustrated by Brian Bolland, Batman: The Killing Joke is a must have for all comic collectors.
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Reviewed in the United States on November 5, 2009