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Description
Mouse AGR2 ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, 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, avoiding repeated freeze-thaw cycles. 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 anterior gradient 2 (AGR2) capture antibody. 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 anterior gradient 2 (AGR2) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Anterior Gradient 2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Anterior gradient protein 2 (AGR-2), also known as secreted cement gland protein XAG-2 homolog, is encoded by the AGR2 gene. AGR2 is located on chromosome 7p21, a region frequently subject to genetic alterations. It is a protein disulfide isomerase with a CXXS active domain motif for both oxidation and reduction reactions. It forms mixed disulfides in substrates, such as intestinal mucins. It interacts with MUC2 through its thiocyanate-like domain, forming heterodisulfide bonds with cysteine residues in the protein. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.2 ★★★★★
Based on 13 reviews
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Product Reviews
★★★★★ 5
Slave Trade was not only a White Man's Affair
Format: Kindle
The book is an excellent read particularly in today's climate. Why 53% of white women would put a vile man in office is explained in the pages of this book. White women held positions of power in the America slave trade, a fact overlooked in history. These southern bell's represented as the gold standard of woman hood in the antebellum south were anything but, and they for the most part showed as much, business savvy as down right cruelty in the slave trade. They benefited in every conceivable way from this free labor market. They were no advocates for the kind humane treatment of slaves. In many cases they were as vicious as their counterpart and just as committed to a keeping Blacks marred in the system of bondage. They are in most cases depicted as silent partners and where that might be the case many white women had full command and knowledge of the value of a slave they invested in and they wanted a hefty return. In fact they used every means on the table to keep these black, men, women and children bound to their wealth creation. These co-conspirators had more than a hand in the cookie jar, they enjoyed the power and did not hesitate to support the maintenance of this inhumane institution.
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Reviewed in the United States on April 24, 2019
★★★★★ 5
Excellent book, not only for lawyers or law students
To begin with, I am neither a lawyer, a law student, nor even a prospective law student. I am, however, someone who has always been interested in the law, primarily for two reasons. First, the law is the principal formal means a society uses to try to resolve conflict among the society's members. Second, and closely related to the first reason, the sum shape, both of content and procedure of the law, is an expression of exactly what a society's values are and the goals a society sets for itself or the standards by which it ideally wishes to be judged.
I found this book to be excellent, informative, well written, and even at parts entertaining. Although meant as a guide for law students to use to prepare for the strenuous exams that are associated with each course they will take in law school, the book provides much, much more, and hence my belief that it can profitably be read by a far larger readership than its ostensible audience. One of the key elements stressed throughout, and exemplified by numerous enlightening examples, is that there usually is no one correct answer to any given legal question. Arguments can be made on at least two sides of any matter based upon, for example, a "plain reading" of the text of a relevant law and the reasonably understandable intent of those who made the law (e.g., a legislature). The authors bring out clearly such sources of legal precedent as laws, government regulations, individual case law decisions by judges, common law, government policy, and specific codes (e.g., the Uniform Commercial Code, or UCC) and show how differing results to a case can readily come about based upon arguments using the different sources to bolster respective cases.
In reality, although by minimal definition a book designed, as said above, to prepare for the taking of law school tests, the book actually also is a good guideline on how to think (not necessarily what to think) about many larger issues in society, including politics and policy issues of all sorts.
Finally, the first two thirds of the book discuss ways to think about the wide range of questions that can be posed to aspiring lawyers and introduces the reader to understanding such distinctions as "forks in the law" and "forks in the facts" (a quite useful distinction to keep in mind). The final part of the book provides solid test taking strategies that are applicable to a wide range of academic testing (e.g., answer the question the professor actually asked and avoid wasting time or effort on ancillary matters not really germane to helping to resolve the issue.) Although some of these may seem obvious once read, the tips are the type of thing that, under pressure of exams, many students often forget to apply.
In sum, I highly recommend this book to those interested in life in the modern world.
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Reviewed in the United States on August 29, 2015
★★★★★ 5
Must read for 1L or Incoming Law Student! So helpful!!!
Format: Paperback
Any 1L or incoming law student needs to read this book! So, I took the BARBI Law Preview before law school began to get an overview of what law school was like and a heads up on how to do things. During this program I had read just two chapters of the book- and these two chapters alone put me in a crucial mind frame to understand the importance of what your professors are looking for. It is not just about distinguishing the right issues and facts, because there is truly no such thing, but distinguishing both sides of an issue, and of course you have to read the book to get more info, but I feel like it has helped me understand what success sounds like in exams. I am only going into my third week of 1L, but I can tell the book has given me a leg up. I recommend that you read this book before you start, or in the first two weeks (though you'll be burdened with a lot of reading then- so before is best) so you can get into the mindset, instead of doing it right before exams and feeling like you have to rewire your brain to everything you thought you understood.
I guess I'll have to update you guys once I see my exams, but so far so good!
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Reviewed in the United States on September 4, 2015
★★★★★ 4
Must-Have for Law Students
Format: Paperback
This is a good book. A lot of the trouble with law school exams is law professors are notoriously bad teachers, and these bad teachers write bad exams. Granted, this is a worst-case scenario, but if you've been to law school for more than one semester, there's a good chance that at least one of your professors has utterly bamboozled you into how he/she wants the final written. So what this book does is give you something of a blueprint and a method of examining fact patterns and exploring the question(s) so that you can simply go into the exam and take it without much fear.
Where the book fails to be of help though, is with the IRAC method. I wholeheartedly agree that IRAC is a too-constrictive method of writing that tends to inhibit most students from really expressing what they know. Law professors largely want a mechanical recitation of rules followed by mechanical analysis, so law students spend hours and hours memorizing rules with the ultimate purpose of using them in an IRAC format. It's absurd, but that's the way it is. And this book simply dismisses the fact that lazy law professors love IRAC for the fact that it gives them a template from which they can read and score exams quickly.
But still, you can construct an IRAC using this method, it just doesn't lend itself seamlessly to it, which is pathetic--not with respect to GTM, but to the teaching and testing methods used by professors. If you don't believe me, and if you haven't already done so, go look at model bar answers from your state and see if they employ a rigid IRAC formula. They don't. And so to me, that's what this book was good for--being able to write bar exam quality answers that leave room for a different writing styles and methods of analysis.
If you're just starting law school, buy this book. If you're already in and still struggling, buy this book. If you're the king or queen of fastidious, multiple, anally retentive headers on your exams, read this book and go look at bar answers.
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Reviewed in the United States on June 22, 2016
★★★★★ 5
The ONLY MARKET-AVAILABLE "ISSUE-IDENTIFICATION" book!!!
Format: Paperback
I purchased it twice: the first time in the law school, but I had misplaced it in the school library & lost it. The second time: while preparing for a BAR exam, I have realized that I material, but I was still missing issues. The book helped. Also, I did not get it on my first read & deeply dissatisfied. But, upon reading the second time & reading it later, I have gotten the point completely. The book helps to formulate what the issues are & you have to understand how to "uncover" the issues prior to formulating the issues. The book helped again.
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Reviewed in the United States on September 15, 2024