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Description
Human UbcH5c 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 utilizes 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 UbcH5c capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and then to yellow by acid. The intensity of the color is positively correlated with the amount of UbcH5c in the sample. Absorbance (OD) is measured at 450 nm using a microplate reader to calculate sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human UbcH5c ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ubiquitin-conjugating enzymes, also known as E2 enzymes and less commonly as ubiquitin-carrying enzymes, perform the second step of the ubiquitination reaction, directing proteins for degradation by the proteasome. The ubiquitination process covalently links ubiquitin (a short protein of 76 amino acids) to a lysine residue on the target protein. Once a protein is tagged with a ubiquitin molecule, additional rounds of ubiquitination form a polyubiquitin chain that is recognized by the proteasome's 19S regulatory particle, triggering ATP-dependent unfolding of the target protein and its entry into the proteasome's 20S core particle, where proteases degrade the target protein into short peptide fragments for cellular recycling. | |||||||||||||||||||||||||||||||||
| 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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4.3 ★★★★★
Based on 1431 reviews
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Product Reviews
★★★★★ 5
Very well written and easy to read.
Format: Paperback
Few people are as qualified as Philip Meyer to write a book on storytelling for lawyers. With a background as a trial lawyer, he has plenty of practical, real-life experience in the courtroom. His approach is not that of an academic giving purely theoretical advice, but that of a seasoned lawyer who knows the ins and outs of the legal profession. His experience as a professor (of both law and writing) has honed his ability to effectively communicate his ideas to a broad audience. Not only is this book helpful for the practicing lawyer, it is also useful and not too complex for the legal neophyte or casual reader.
This book breaks storytelling (narrative) down to its core components and analyzes them one by one. In the process of analyzing each part of a story, Philip Meyer skillfully explores each component with a non-legal example (e.g. movies, books, etc.) before applying it to a legal example (e.g. courtroom proceedings, appellate briefs, closing arguments, etc.) By first analyzing each part of a story (i.e. plot, setting, etc.) from a well-known story that resonates with the reader, he sets a strong foundation before transitioning to a legal story, thus making it easy for the reader to identify and better understand each part of the legal story.
I highly recommend this book to anyone remotely interested in storytelling and persuasion as they relate to the legal profession.
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Reviewed in the United States on November 22, 2016
★★★★★ 4
Must Read for Novice Litigators
Format: Paperback
This book is a great starting point for developing the skill of storytelling for lawyers as was intended by the author. The author gives you the basics for developing the plot, characters, style, setting, and narrative for your trial with excellent examples. The author is a law professor and the book seems geared for the law student or novice lawyers getting into litigation.
I only gave the book 4 out of 5 stars because of a couple of minor problems. However, the chapter on narrative needs further exposition and appears to be written in rushed manner. In addition, the physical binding of the book is of poor quality requiring me to glue the cover back on. Finally, the author missed the point that the lawyer's job is to look at his case as a giant puzzle to be solved and then explained as a story.It is not enough to understand your case but equally imperative that you communicate your case which is best done through the storytelling technique. This is a must read for lawyers getting up to speed on litigation.
For further exposition on legal storytelling for lawyers after reading Meyer's book on Storytelling for Lawyers, I recommend the following:
ABA webinar available with an internet search for "Storytelling for Lawyers"
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Reviewed in the United States on July 25, 2018
★★★★★ 5
Much needed guide to narration in law practice
Format: Paperback
Meyer’s “Storytelling for Lawyers” is an important contribution to the literature on narration in law practice. We know that successful courtroom rhetoric can best be viewed through the prism of storytelling. But the literature does not contain a practical and detailed analysis of the elements of narration as used in law practice—that is, plotting, characterization, point of view, style, and settings in place and time. Meyer’s book fills this gap. It is blessedly free of jargon and full of practical examples of good legal storytelling. But the importance of this book goes well beyond providing practical assistance to litigators. It serves as a much-needed introduction to the principles of narration for teachers and students of literature, creative writing, and popular culture, who have lacked a readable introductory guide to the elements of successful storytelling.
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Reviewed in the United States on February 10, 2014
★★★★★ 5
Recommended for All Lawyers
Format: Paperback
Meyer proves his initial point that much of what lawyers do is storytelling, and he achieves his goal of providing a primer on narrative theory for lawyer-storytellers. The book is sophisticated but written in an engaging way using non-technical language. Examples from legal and literary works abound, and they range from courtroom arguments and appellate briefs on the one hand to an essay by Joan Didion and Vonnegut's "Slaughterhouse Five" on the other. Meyer's favorite stories are found in Hollywood movies, and although he seems unaware of the accomplishment,Meyer provides fresh interpretations of such movies as "HIgh Noon" and"Jaws." I strongly recommend "Storytelling for Lawyers" for all law students, lawyers, and judges.
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Reviewed in the United States on May 7, 2014
★★★★★ 3
Notes on Legal Style by a Law Professor and Experienced Lawyer.
Format: Kindle
BOOK REVIEW: MEYER, Philip N., Storytelling for Lawyers
ISBN: 978-0-19-5396638
Read June, 13th-27th, 2017.
This book discusses storytelling tools by presenting a series of examples of good storytelling, both in legal settings and in literary works and movies. If theoretical explanations are sometimes a bit dry, the frequent quoting of practical examples conveys fluidity and speed to the book.
After an introduction presenting lawyers as storytellers, it deals with the roles played in storytelling by Plots (chapters 2 and 3); Character (4 and 5); Voice, Perspective, Details and Images, and Rhytm and Speed (which relate to Scene and Summary) (chapter 6); Place or Story Environment (chapter 7) and Narrative Time.
Focusing maybe too narrowly on legal storytelling before American juries, plot is almost equated with melodrama. Films like Jaws and High Noon are extensively discussed, as Gerry Spence’s Closing Argument on Behalf of Karen Silkwood. The chapters on character offer interesting insights on character classification (“round” characters, with psychological depth, prone to suffer transformation as the story evolves, vs. “flat” ones), while discussing the tools for telling how a character is, as opposed to simply showing the psychological nature of each character’s character through dialogue or the actions the character performs. Examples include Tobias Wolff’s This Boy’s Life and Jeremiah Donovan’s Closing Arguments on Behalf of Louis Failla, in a 13-week trial the Author could scrupulously attend in person.
Discussions on Voice, Perspective, Details and Images, Scene and Summary, criticize the basic assumptions of the neutrality of lawyers’ voices, exemplifies how to manage details to suggest ideas and emotions, draw on the distinction between showing and telling, and offers interesting insights into the narrative theory’s concept of stretch (the slowing of the narrative rhythm in relation to the narrated story’s). Environment depiction storytelling tools deals with Joan Didion’s The White Album and the Judicial Opinion in a Rape Case, quoting also from W. G. Sebald’s The Emigrants and the Petition Briefs in Reck v. Ragen and Miranda v. Arizona. Further examples are Kathryn Harrison’s While They Slept and the Petitioner’s Brief in Eddings v. Oklahoma.
Finally, the chapter on Narrative Time draws on Kurt Vonnegut’s Slaughterhouse Five and explores time, rhythm or speed, discussing more deeply stretch and the relation of time of the narrative itself with the time of the facts dealt with in the narrative. Chronology is discussed and criticized; Analepsis or Flashback is didactically explained and exemplified, both in general storytelling theory and in its legal use; the same holds for Prolepsis (Flash-forward) and Ellipsis (the intentional omission of a part of the narrative, often with the purpose of emphasizing the omitted event. Pacing and Rhythm are discussed in more lenght, with the caveat - repeated somewhat throughout the book - that legal stories are often left unfinished by the lawyer, in order to allow the jurors or judges fill the end with their decision.
The Author remarks his purpose was to suggest possible tools and ways of dealing with problems which arise in legal storytelling, and he delivers what he promises.
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Reviewed in the United States on June 27, 2017
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