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Description
Human TFAM 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 Transcription Factor A, Mitochondrial (TFAM) 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 Transcription Factor A, Mitochondrial (TFAM) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Transcription Factor A, Mitochondrial ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Mitochondrial transcription factor A (TFAM) is a protein encoded by the TFAM gene. This gene encodes a mitochondrial transcription factor that is a key activator of mitochondrial transcription and a participant in mitochondrial genome replication. It binds to mitochondrial promoter DNA to facilitate mitochondrial genome transcription. It is a double-box, high-mobility group DNA binding and bending protein. This bending function is important for mitochondrial transcription initiation in mammals, but not in its yeast homolog, Abf2. | |||||||||||||||||||||||||||||||||
| 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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4.4 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 5
If you can only afford one craft book on Dialogue, this one is it.
Format: Hardcover
The art of dialogue. How does a writer get it just right to be effective, yet original, dramatic but not too dramatic, captivating and satisfying, and most important of all convincing? The thrust of this book is how McKee spends time on characterizations and the art of the subtext—vital to thrilling and effective dialogue. McKee explains how subtext works, the thinking behind it, shows you its most effective moments, why it works well or poorly, and gives you the tools to make it work. The result: amazingly clear insight. No kidding, if you want to fully understand subtext in dialogue and sharpen your skills, this is the writing book to get.
Conflict in dialogue, turning points, even sentence designs techniques. McKee describes the “suspense sentence” and the “periodic sentence.” Because prose is a natural medium for storytelling, you will learn how Charles Dickens used ‘counter pointing exposition’ and its effectiveness for the reader. I hadn’t seen this kind of hook in writing before, hadn’t heard this term before. Very powerful technique; of course none of us writes like Dickens, but what an example on how to swoop the reader in.
What’s one of the takeaways in Dialogue by Robert McKee? “Quality storytelling inspires quality dialogue.” Which inspires a read of McKee’s other remarkable craft book “Story”—a great companion read as both books belong on any serious writer’s bookshelf.
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Reviewed in the United States on July 28, 2016
★★★★★ 5
It's All Right Here. All of it.
Format: Hardcover
I shouldn't even be writing this review because doing so only creates my competition. I'm writing it for Robert McKee out of respect and love for him as one of the best instructors I've never met. I have both STORY and now, DIALOGUE in hardcover, on my Kindle, and the audio version (he narrates his books) in my headphones. I read it, I listen to it and take it with me when I travel. Sometimes I listen and read at the same time. Most nights I listen to the audio version in my sleep. In other words, I eat, sleep and breathe this book. I did the same thing with Story, his other book. I may go to my grave never fully comprehending the vast wealth of knowledge contained in these two books. That's OK. What I can tell you is this. With nothing but STORY as my guide, my very first screenplay took seven months and thirty-five drafts from start to finish. But. That screenplay became a Hallmark Movies and Mysteries feature film. The producers liked it so much they gave me another assignment. That one took six weeks, and they bought the first draft. I'm now working on my fifth script; this one is in the six-figure category, with five figures upfront just for the Treatment. And I owe it all to everything I've learned from studying Robert McKee, supplemented by what I learned from everyone else. Over the past thirty years, I’ve studied with forty plus instructors and highlighted hundreds of books and listened to dozens of recorded seminars. All that information is summed up and thoroughly explained in Robert McKee's two books STORY and now DIALOGUE. I won't live long enough to absorb everything he teaches. And I still study two hours a day as a warm up for my writing. I’d recommend Aaron Sorkin, Warner Hertzog, William Goldman (both Sorkin and McKee say he's the greatest), Blake Synder, Chris Vogler, Michael Hauge, William Akers, and anybody else you can find who’s willing to share their knowledge. Because you never know when a concept you didn’t realize you didn’t understand or needed is going to show up. Especially when presented from a different perspective. Having said that, if you are serious, and I mean dead serious, about becoming a working screenwriter, or any other kind of fiction writer for that matter, then you have no choice but to study McKee like your literary life depends on it. Buy the hardcover, buy the Kindle version, and buy the audio version of DIALOGUE and STORY. And supplement these two works with any other material that speaks to you. If you do this, you will become a first class screenwriter or novelist or playwright, because all three genres are only different ways of presenting a Story. If you can’t commit to this, unless you’re a genius or prodigy, you’re wasting valuable time which could be spent following your true life calling. But if your heart’s desire is to become a working writer, then sooner or later you’ll have to know everything in McKee’s two books. So, you might as well bite the bullet and jump in head first. It's all right here in STORY and DIALOGUE. All of it. Thank you, Mr. McKee. You, sir, changed my life.-- Jimmy Hager
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Reviewed in the United States on September 21, 2016
★★★★★ 5
A book on dialogue should be experienced first as a book on tape
Format: Audio CD
I think of this more as a great master class lecture. Dialogue should be seemingly simple (we all talk), but McKee defines its essence and differences for prose, stage and cinema. The bulk is narrated by McKee, but the scene examples are read by voice actors and they do quite well. Even the roots of the English language are examined in order to make better decisions on your character's particular use of words. After listening the 10 hours twice while commuting, I finally picked up the book and read it. The book on tape is a better way to initially absorb the material, while the actual book helps to clarify the info. A must for all writers, especially screenwriters.
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Reviewed in the United States on August 30, 2018
★★★★★ 4
Helpful, but not as good as "Story" by same author, and it disses certain genres
Format: Hardcover
This book contains a lot of helpful information on how to write dialogue. It's dense with dialogue analysis and insights, tough to take in by just reading it through once. But it is helpful.
McKee covers the three dialogue tiers (said, unsaid, unsayable) as well as how dialogue ties into story turning points and scene conflict type. I still have lots of practice ahead of me to figure out how best to do this in my story. I will definitely use his advice as a guide. He understands dialogue at a much deeper level than I do.
However, many of McKee's dialogue examples did not speak to me. While I liked reading the dialogue examples for Breaking Bad, 30 Rock, The Sopranos, Frasier, A Raisin in the Sun, and The Great Gatsby, and agreed they were good, I disliked the dialogue from Shakespeare, Elmore Leonard, Sideways, Fraulein Else, and Lost in Translation. McKee says fine dialogue turns the reader/audience into a mind reader; I guess I'm not interested in movies which expect me to be as much of a mind reader as those latter examples did. I totally missed the subtext of the dialogue in those until he explained it to me as an aside. And that's after I already saw most of those movies!
If I have to guess what every character means with every line, that's too much work and too little entertainment for me. Maybe mystery lovers liked the dialogue in "Lost in Translation"; I'm not a mystery lover. McKee quoted one novelist as saying that the crux of good writing is to, "Make em laugh, make em cry, make em wait." Lost In Translation and its dialogue did none of that for me. The subtext was so confusing and subtle that I lost interest in the movie. I can't even remember what it was about anymore, only that it won some award and I had no clue why.
McKee says that with rare exceptions, a scene should never be outwardly and entirely about what it seems to be about. Dialogue should imply, not explain, its subtext. An ever-present subtext is the guiding principle of realism.
Nonrealism, on the other hand, employs on-the-nose dialogue in all its genres and subgenres: myth and fairytale, science fiction and time travel, animation, the musical, the supernatural, Theatre of the Absurd, action/adventure, farce, horror, allegory, magical realism, postmodernism, dieselpunk retrofuturism, and the like. It's a bit unclear how, if at all, anyone writing in any of these "nonreal" genres should take his dialogue advice. It seems to me that even sci fi scenes need some good dialogue with subtext to be engaging.
With McKee, all the accolades go to what is implied and unsaid over what is said. I agree that subtext matters, but for me, he's out of proportion with how much it matters to most people and how hard audiences are willing to work to discover the intended subtext. Also, memorable spoken character lines can elevate movie themes and characterization like nothing else.
In the end, I think this book is geared more toward writers who want other advanced writers as their audience rather than the average reader or movie watcher. And McKee admits it is definitely not geared toward sci fi, fairytales/myths, action/adventure, horror or allegory. It's almost as if he's saying those genres can't have excellent dialogue. I disagree.
But it was still a helpful book to read, and one I will be thinking about and trying to more fully understand for a long time. McKee understands how character's subconscious drives can deepen what they say or avoid saying, and how dialogue interacts with many other aspects of a story to make it all work together.
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Reviewed in the United States on December 12, 2019
★★★★★ 5
Amazing.
Format: Kindle
Just like a good movie, the first 10 pages = mind blown. Wow, such really, really good material here. If you're new, this will help you a ton. If you're experienced, this book will help you realize WHY great dialogue is so great, enabling you to create the magic again and again. I love how McKee covers several medias ( screen, theater, novel ) but still stays true and clear on the concept. A virtual masterclass on the subject. One of the best screenwriting books out there, and Yes, it's well worth all the hype.
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Reviewed in the United States on April 18, 2017
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