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Description
Mouse AGC 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: 1. 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: 1. 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. 2. 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. 3. 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. 4. 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. Preparation of standard gradient working solution: Add 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 5000pg/mL). Then dilute to the following concentrations: 5000pg/mL, 2500pg/mL, 1250pg/mL, 625pg/mL, 312.5pg/mL, 156.25pg/mL, 78.125pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 5000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 2500pg/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 Aggrecan (AGC) 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 Aggrecan (AGC) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Aggrecan ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Aggrecan, also known as cartilage-specific proteoglycan core protein (CSPCP) or chondroitin sulfate proteoglycan 1, is a protein encoded by the ACAN gene. This gene is a member of the clockican (chondroitin sulfate proteoglycan) family. The encoded protein is a component of the extracellular matrix in cartilage tissue, which supports cartilage compression. Aggrecan is a proteoglycan, or a protein modified with large carbohydrates. This protein is 2,316 amino acids long and, due to alternative splicing, can be expressed as multiple isoforms. It is also a high-molecular-weight proteoglycan. It exhibits a bottle-shaped structure in which chondroitin sulfate and keratan sulfate glycosaminoglycan (GAG) chains are attached to an extended protein core. Aggrecan has a molecular mass greater than 2,500 kDa. The core protein (approximately 300 kDa) has approximately 100 GAG chains attached to it. Aggrecan consists of two globular domains at the N-terminus (G1 and G2) and a single globular domain at the C-terminus (G3), separated by a large extended domain (CS) that is heavily modified with GAGs. These two major modifications are themselves arranged into distinct domains: a chondroitin sulfate domain and a keratan sulfate domain. Aggrecan also plays a crucial role in organizing the extracellular space between neurons in the brain. Through interactions with linker proteins and tenascins, aggrecan binds to hyaluronic acid, forming large aggregated complexes on the cell surface. | |||||||||||||||||||||||||||||||||
| 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 | 78.1-5000pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates and other biological fluids |
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4.6 ★★★★★
Based on 5 reviews
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Product Reviews
★★★★★ 5
Truly "essential" for any letterer, whether casual or serious about the profession!
Format: Paperback
When I first got this book, I was a bit disappointed. I thought it was going to be detailing lettering by hand.
It didn't.
But guess what? It's better for it.
As the author, Nate Peikos, demonstrates, the bulk of lettering now is done digitally and with good reason. So not only is this book incredibly inspiring for any graphic artist / letterer -- with its many examples of great lettering; its demonstration of useful techniques -- the author is so open and honest in his writing. While reading it, you don't feel that he's putting the job of lettering out of the reader's reach. In fact, he's putting it right within reach through a humility that is coupled with his exceptional skills. If you're an up-and-coming letterer, someone on the outskirts of lettering wanting to delve into it more professional (like myself), or even if you've had some work doing lettering, there is something for each type in this book. It's a book that I'll cherish and reference frequently, especially with all the examples that just ooze inspiration.
My only regret is that I didn't know about some of these techniques a few years ago! But, nevertheless, I started implementing some of the techniques the first day after reading just a few pages. It reinvigorated my own desire to become a better and even semi-professional letterer, a very under-appreciated profession.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 5, 2021
★★★★★ 4
A great overall book, with one critical omission.
Format: Paperback
This is a great book to illuminate the mysterious art of comicbook lettering. It goes deep into the practical details and theory required to make a graphic novel, manga, or superhero comic with the look and feel of a professional product. But comicbook lettering is actually more than a purely aesthetic art form, it plays a critical role in conveying meaning, emotional mood, and tone of voice. Along with the artwork and panel composition, the layout of the lettering and dialogue balloons is also critical to help guide the reader's eyes naturally from panel to panel.
If you're curious about how graphic design for visual narrative works, this is an interesting and inspiring book.
My only criticism is the conspicuous omission of perhaps the one topic this author is uniquely qualified to illuminate, and that is: the actual creation of the fonts themselves. I've admired Nate's Blambot comic fonts for a long time (and have purchased licenses for some of them), so I was looking forward to at least getting an overview of how he approaches this seemingly critical aspect of comicbook lettering. But no. He doesn't go into it even slightly, which is a shame, because this would have been the perfect book on the topic if he had. I understand that font design is a huge topic unto itself, but was disappointed that in a book of this size, there weren't even a few pages dedicated to discussing the process of making fonts for comicbooks. Literally anyone reading this book would want to know that information.
Having said that, it's a great book, and I still enjoyed it very much. You'll probably be surprised by how much more there is to learn about this topic than you may have thought. Reading it will deepen your appreciation for an art form that, like a great movie score, is basically invisible when done very well, yet amplifies the emotional impact of each contributing discipline, and profoundly contributes to the enjoyment of the final experience.
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Reviewed in the United States on September 22, 2022
★★★★★ 5
A Lettering Masterclass
Format: Paperback
Nate Piekos is a rockstar. If you want it learn how to letter comics, this is the first thing I would buy. It’s not a fancy book, just practical step-by-step how to do a craft right.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 30, 2026
★★★★★ 5
Title says it all: ESSENTIAL!
Format: Paperback
This is a FANTASTIC book! The information may seem overwhelming at first, but it is easy to understand.
As some reviewers said, it does not spend much time on hand lettering (I assume this is due to the industry going more digital as time goes on). Those who want more information on hand lettering may have to seek help elsewhere. It is mentioned VERY briefly in the beginning, I believe.
It includes: step by step instructions on creating speech bubbles, tips on font creations, sentence formatting, page borders, splash/double splash page construction, creating templates that save you time in the long run, making logos, sound effects and much more.
This book is a great launching point for anyone looking to start their lettering career or even for anyone that wants to gain an appreciation for an intergral and often overlooked part of Comic books!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 28, 2022
★★★★★ 5
One of the Best "Making Comics" Books Out There
Format: Kindle
Honestly, one of the best books on making comics that I've read. So well done, insightful, well-organized, and easily referential. If you're looking for the basic principles of lettering, look no further than this.
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Reviewed in the United States on June 22, 2025