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Description
Human TRA2b 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 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, biotinylated detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Transformer-2 protein homolog beta (TRA2b). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of Transformer-2 protein homolog beta (TRA2b) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Transformer-2 protein homolog beta ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Transforming factor 2 beta (TRA2b), also known as splicing factor arginine/serine-rich 10 (SFRS10), is a protein encoded by the TRA2b gene. This gene encodes a nuclear protein that functions as a sequence-specific serine/arginine splicing factor, playing a role in mRNA processing, splicing patterns, and gene expression. It activates splicing of MAPT/Tau exon 10. It alters pre-mRNA splicing patterns by antagonizing the actions of splicing regulators such as RBMX. It binds to the AG-rich SE2 domain of SMN exon 7 RNA. It binds to pre-mRNA. Diseases associated with it include frontotemporal dementia and spinal muscular atrophy. It has been shown to interact with RBMX. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.6 ★★★★★
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Product Reviews
★★★★★ 4
Informative
Format: Paperback
This book was one of several college informaion books I checked out from our local library. This one was the best - informative, objective and not-too-lengthy or not-too-short for each important element of college selection and application processes. So, I decided to buy one to keep at home. I wish the paper is thicker and better quality. Whenever I use highlighter, it got through its back-page.
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Reviewed in the United States on June 19, 2013
★★★★★ 5
Excellent advice
Format: Paperback
I've been working in College Student Affairs for almost 10 years (admissions & academic advising.) I bought this as a gift for my godson, who was a sophomore in high school at the time. I chose this book because it didn't put pressure on kids to pick the big name school with the highest price tag they were accepted to. It gave good advice bout finding out and figuring out what schools would be a good "fit" for them. It also had some good advice about taking & prepping for the SATs - which made it a good choice fod my godson even though he is a couple years away from filling out his applications!
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Reviewed in the United States on January 18, 2012
★★★★★ 5
Highly Recommended
Format: Paperback
Superb book that eased my parental anxieties and has proven very helpful to my college-applicant daughter. Even though it was published in 2011, it is *not* dated (something I worried about). Sure, it will probably need an update by 2020 or so, but most of the info is not very time-sensitive to begin with.
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Reviewed in the United States on February 5, 2017
★★★★★ 3
Good place to start
Format: Paperback
I am an independent college adviser based in California and I recommend this book to my clients but only at the very beginning of the process (mostly to the parents of rising high school freshmen and sophomores).
This book is a good place to start especially for many of the foreign educated or immigrant families, who are new to the US College Admission process. The parents do tell us that the book loses its effectiveness once they gain knowledge of the process and/or they begin working one-on-one with an independent and/or become more conscious about meeting the high school counselors early in the process.
I personally like the fact that once this book is overviewed by the families, we can spend more time addressing specific concerns and giving more customized advice. As a result we only need quarterly meetings and these meetings are extremely targeted.
The problem with this book though is it reads like a huge information session by colleges. Those of you who have attended some of the information sessions will know what I mean. The insights provided by the deans and admission officers of various colleges sound somewhat like a "sales pitch" at times and at other times are misleading. I also recommend my students to keep their knowledge up to date by referring to various free resources such as Nancy Griesemer's educational articles in Washington Examiner Online, or the Choice Blog. I spend considerable time volunteering my services at a high school in a mixed/low income neighborhoods; I wish the price had been more reasonable considering that this is a basic book.
Lastly as members of NACAC (National Association of College Admission Counseling), all of us in the admissions field (College Admission Officers, High School Counselors, and Independent Counselors) subscribe to certain codes of ethics and one of the Statements of Good Practice says this about Fairness and Equity: We believe our members have a responsibility to treat one another and students in a fundamentally fair and equitable manner. Unfortunately, Ms. Mamlet and Ms. VanDeVelde do a great disservice by being disrespectful to a large community of Independent College Advisers, many of whom are needed when the high school counselors are juggling with case loads of 200-300 students each and are unable to keep up with the changing scene of college admissions (either due to the lack of funding or time). Gone are the days when people went to some "quack-independents" who promised admissions to Ivy Leagues--sure there are some of those but if families are careful in their research, they will soon find out that these Independents do not belong to any authentic professional organizations such as the NACAC, HECA or IECA. Most students who come to the authentic professionals are motivated to find their right fit college, explore their own strengths and weaknesses and find the best match for their talents, interests and financial situation. Most independents offer services that are reasonably priced. If Ms. Mamlet and VanDeVelde show more commitment to researching folks on the other side of the spectrum, I am sure that their book will be more enlightening.
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Reviewed in the United States on August 18, 2013
★★★★★ 5
Best College Info Book Ever!
Format: Paperback
I have read dozens of college-related books in the past 4 years, and this one ranks the highest by far. Written by industry experts and the authors got a lot of top talent to contribute. Full of real-life advice and useful information that is actually true (I fact-checked this against some other info).
I read parts of it out loud to my junior on the plane en route to a college tour, and she actually listened! Humor is always appreciated, and these authors use it without going overboard.
Great information, well-presented. A must-read!
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Reviewed in the United States on February 22, 2013