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Description
Human PCNA 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: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled 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 freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 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 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves 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 Proliferating Cell Nuclear Antigen (PCNA) 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 Proliferating Cell Nuclear Antigen (PCNA) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Proliferating Cell Nuclear Antigen | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Proliferating cell nuclear antigen (PCNA) is a DNA clamp that acts as a promoter of DNA polymerase delta in eukaryotic cells and is essential for replication. PCNA is a homotrimer that achieves its activity by encircling DNA, where it serves as a scaffold to recruit proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics. The protein encoded by this gene is present in the cell nucleus and serves as a cofactor for DNA polymerase delta. As a homotrimer, the encoded protein helps increase the efficiency of leading-strand synthesis during DNA replication. In response to DNA damage, the protein is ubiquitinated and participates in the RAD6-dependent DNA repair pathway. Two transcript variants encoding the same protein have been identified. Pseudogenes of this gene have been described on chromosomes 4 and X. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.8 ★★★★★
Based on 23 reviews
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Product Reviews
★★★★★ 5
Will reading Wired for Story really make you smarter?
Format: Paperback
In my 36th year as a would-be and penniless writer, I found myself exiled to a dark rough and tumble city in the Far West, guns blazing as a steely-eyed wordslinger for hire. But then one day I stumbled upon Lisa Cron's book Wired for Story.
The book's title had my curiosity. A few sample pages later grabbed my attention and has held it ever since. But the price wasn't right for a poor, humble English teacher living in China upon a Chinese salary. I had bills to pay, a mistress to please, and habits to feed. It seemed to me that Amazon.com was colluding with other dark powers to suck humanity dry; why else would they charge more for a digital book than its paper copy?
But then I heard ghostly voices, the cinematic intonations of Morpheus telling me to choose between the red and blue pill; Obiwan Kenobi, "Use the Force"; Nike commercials, "Just Do It!"; and other such shadows flickering upon the wall of my TV room. Even this very particular retail website seemed to whisper across all the vastness of cyberspace, reminding me of my destiny via a personalized showcase of products, that I was not just born to buy...
So I added it to my cart. About a download and two chapters later I found that I was still happy after the post-purchase buzz ran its course.
This book should be required reading for all writers - and anybody else seeking an inoculation against the raging pandemic of competing narratives spewed out from marketers, pundits, prophets, and others posing as guardians of the truth - most of whom seem to be more enraptured than enlightened.
For writers though, Wired for Story is quite different from other "how to" books, as Lisa Cron approaches the craft of storytelling from a neuroscientific point of view. She makes the case that writers aren't just entertainers: they are some of most powerful shakers and shapers of human perception.
So if storytellers are like snake oil salesmen, then what is the difference? Both seem to be highly skilled in crafting story, using imagery, and evoking emotions, memories, desires. The difference is all about marketing. Salesmen claim to have knowledge, skills, and expertise, that they, and they alone have whatever it takes to get the facts right and fix things. They market their brands cloaked in story, as if they have a monopoly on truth, or at least the can-do spirit and problem-solving experience needed to improve the economy, save the world, whatever. It doesn't matter that time and time again reality proves them wrong; they will always have another story to spin.
The difference between those who would use the power of story to express themselves versus those who would use it for personal gain is, perhaps, a fine red line marking the shadowy borders of between ethics and morality. Storytellers differ because they use words to hook audiences and manipulate a willing reader's central nervous system. They make no claims to knowledge or expertise.
Indeed, fiction writers will be first to emphasize their work is fictional, and not based on any real life events or people. Their best writing leaves readers thinking, questioning, minds opening, empathizing, expanding their worldviews, the list goes on almost ad infinitum. Storytellers speak for themselves and let audiences think for themselves; pundits speak for others and tell audiences what to think.
What's more, the art and craft of story, as well as the talent and hard time in solitary confinement required for their honing, is estimated to take an average storyteller at least 1,000,000 words or 10,000 hours - not including all the reading, language arts development, and life experience necessary to get to a point one needs to seriously embark on such a ludicrous and un-economical vocation.
This means that fiction writers who risk everything for dubious prospects of financial reward must have something else driving them - and a good day job. A presidential candidate though, who has genuinely done the time, and crafts speeches with the skill of a poet or bard, should hypothetically have the critical thinking background, moral authority, and empathy to be a great leader. But in the final analysis, actions contradict words; their ability to spin tales proves the old universal theme that the pen is mightier than the sword.
Now when I finish Wired for Story sometime this week, I will be one step further on this endless quest to actually sell stories for a living (i.e. stories fit for the fiction aisle of an actual bookstore, not a review for an online retailer). Until then, I'm probably just a hypocrite acting as if a single book alone makes a smarter man, when in fact I know little of anything (which is why I became a writer in the first place) -- or maintaining such a humble pretense. But I don't know myself well enough to be certain. That kind of exploration would be a whole other story - but it would be unsafe to say that I lived happily ever after reading this book.
The End
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Reviewed in the United States on October 11, 2012
★★★★★ 4
A Cheeky Novel about writing cheeky novels
Format: Paperback
Wired for Story is a book full of solid, albeit basic, advice for story tellers, and in particular writers trying to develop their craft. It succeeds in some areas while falls down in others, however I found it solid and well written overall.
Where it succeeds is in its brevity and clarity. The author gets right to the point and even provides short lists at times of what to do and how to do it. Her writing is also peppered with cheeky humor which is humorous in a thats-almost-funny kind of way that is refreshing compared to the textbook style adopted by many how-to-write books.
Where the book fell down, for me, was in its limited examples and scope. It really felt like the author was addressing romance writers, for the most part. I could think of a few counter examples to some of her rules, although one would have to leave the romance genre for those to work. But for a 230 page book, that is minor discrepancy.
The second thing I noticed, which has already been pointed out by others, is that the brain science was limited. In fact, it was mostly only refereed to in footnote. Personally, that was fine with me, but I could see why it made others feel misled (it is in the title, after all).
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Reviewed in the United States on December 5, 2014
★★★★★ 5
Unfortunately yet another one to add on the pile of writing books!
Format: Paperback
I say 'unfortunately another good book on writing' since I have been reading many books on the topic and each time I think I have read it all by now, pfff, but then another one like this comes along. But hey, it is fun to read about writing and especially if you get something useful and new out of it. Always worth the money.
Why the accolades for this book?
All the information on characters, plot, details etc, how they work and why they are important, is not new. You find them in many reading books, especially from Reader's and Writer's Digest series. I would especially recommend the books on plot and structure by James Scott Bell.
Yet this book Wired For Story is not redundant, since it is unique in both the focus on the familiar subjects like what story is, the importance of details, details versus generics, how events affect a character etc. The approach of using brain science shows in a compelling way why all the stuff in the book (and for that matter, the classic theory on writing) is there for a reason, since it has evolved out of how we humans seem to perceive a story. Interesting though, a lot of rules like 'write about what you know' are shown to be myths.
Of course, in the end it is you as a reader that has to decide what works for you, but you will find new things here and that is always good.
If you are thinking about writing a story, this book can give you a very sharp focus on what to think about before you start. Or, like me, when you have a draft, and you want to get to the 'real story' behind it to come forward, this work gives you a very transparent way to get better clues as to what stays and what has to go.
It is also entertaining and easy to read, so, all I can say is, yet again a book on writing you cannot afford to miss!
I just added some extra information: I loved the humor and wisdom of the writer. She has worked for years reading manuscripts and she does know her stuff, and I liked her tone, which was both realistic but also fun! How many times have you read a reference work and actually had to laugh? But this book was also profound on many story issues. Many common writing rules are, as I said before, shown to be myths and more importantly, why this is so. I have read for instance so often that you should have some sensory info on every page, but, as I thought myself and I am not the only one: although this makes things more lively, it should only be there if the story would dictate it! The book has many more things like these.
I have compiled a collection of notes I took from all the many many books on writing so I measure new books on writing in how much text I copied and pasted to look at again and this book has given me lots, so 5 stars and I hope we will see more of this author again!
I also recommend the book by James Scott Bell on plot and structure which you can find here
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Reviewed in the United States on October 28, 2012
★★★★★ 5
Great book
Format: Kindle
I have been an author for decades and published 4 books. I've been an editor for over a decade and this book is perfect for seasoned and new writers. It certainly made me take a new, long look at the latest book I'm busy with. I think it's great for getting one focused on the important points. I loved it. There wasn't too much about how the brain works to bore one or make it feel like a technical book on neuroscience. I found her writing style amusing and entertaining. I will definitely recommend it to all my clients and writing friends. There was nothing in the book I didn't know, but this book forces one to focus on the bigger picture. It doesn't matter how accomplished one is as a writer, one can always learn more. And I certainly did with this book. So, thank you, Ms. Cron.
I saw the negative three or fewer star comments and can only think the commentators didn't see what the author was actually trying to convey. It's a classic lesson in the KISS method (keep it simple stupid) and I loved it.
This isn't a full-on, in-depth analysis of every part of the craft of writing, as many seemed to expect, that would take 12 full books or more. This is also not only for beginners as some suggest. To them I would say, don't let hubris cloud your ability to write better--think beyond that--read the book again. I think the book serves as an excellent reference to keep us on track when we lose the plot, so to speak.
Seasoned writers can get quite jaded and write almost on auto-pilot at times--not good. Thank you, Ms. Cron, for putting me back on track and making me excited to re-write and re-edit my latest book, my first YA fantasy, for the zillionth time. I was out of my comfort zone with this book and feeling a bit overwhelmed and unsure. Wired for Story has me fired up to go forth and do this.
I highly recommend this book, especially for seasoned writers.
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Reviewed in the United States on January 26, 2018
★★★★★ 5
Quality writing information
Format: Paperback
A book that was fun to read and informative. I feel as though I have a better understanding of writing fiction now.
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Reviewed in the United States on February 11, 2026