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Description
Rabbit MYO ELISA KitProduct Specification Usage I. Sample Collection, Preparation, and Storage 1. Serum: After placing whole blood samples at room temperature for 2 hours or at 4C overnight, centrifuge at 1000g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen free, and endotoxin free. Store at 20C or 80C and avoid repeated freezing and thawing. 2. Plasma: Within 30 minutes of collection, centrifuge at 1000g for 15
Product Specification
| Usage | I. Sample Collection, Preparation, and Storage 1. Serum: After placing whole blood samples at room temperature for 2 hours or at 4°C overnight, centrifuge at 1000×g for 20 minutes. Remove the supernatant for testing. Blood collection tubes should be disposable, pyrogen-free, and endotoxin-free. Store at -20°C or -80°C and avoid repeated freezing and thawing. 2. Plasma: Within 30 minutes of collection, centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing. EDTA-Na2 is recommended as the anticoagulant to avoid hemolysis or hyperlipidemia. Store at -20°C or -80°C and avoid repeated freezing and thawing. 3. Tissue Homogenization: Take an appropriate amount of tissue and wash it in pre-chilled PBS (0.01M, pH 7.0-7.2) to remove blood (lysed red blood cells in the homogenate will affect the measurement results). After weighing, mince the tissue and mix it with the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio; the specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS). Pour the mixture into a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or freeze-thawed repeatedly (keep the sonication in an ice bath and repeat the freeze-thaw cycle twice). Finally, centrifuge the homogenate at 5000×g for 5-10 minutes. Remove the supernatant for analysis. 4. Cell Culture Supernatant: Centrifuge the cell supernatant at 1000×g for 20 minutes to remove impurities and cell debris. Remove the supernatant for testing and store at -20°C or -80°C, but avoid repeated freezing and thawing. 5. Urine: Collect the first morning urine (midstream) or 24-hour urine collection. Centrifuge at 2000×g for 15 minutes, collect the supernatant, and store the sample at -20°C. Avoid repeated freezing and thawing. 6. Saliva: Collect the sample using a saliva sample collection tube, then centrifuge at 1000×g for 15 minutes at 2-8°C. Remove the supernatant for testing, or aliquot and store at -20°C. Avoid repeated freezing and thawing. 7. Other biological samples: Centrifuge at 1000×g for 20 minutes, collect the supernatant, and store at -20°C. Avoid repeated freezing and thawing. Notes: 1. Samples should be clear and transparent, and suspended matter should be removed by centrifugation. Hemolysis of the sample will affect the results, so hemolyzed samples should not be used. 2. Samples can be stored at 4°C if tested within one week of collection. If testing cannot be performed promptly, aliquot the sample into single-use portions and freeze at -20°C (for testing within one month) or -80°C (for testing within three to six months). Avoid repeated freeze-thaw cycles. Bring the sample to room temperature before experimenting. 3. If the concentration of the test substance in your sample is higher than the highest value of the standard, please dilute it appropriately based on the actual situation (it is recommended to conduct a pilot experiment to determine the dilution factor). 2. Pre-Assay Preparation 1. Remove the test kit from the refrigerator 30 minutes in advance and equilibrate to room temperature. 2. Dilute 25 μg of concentrated wash buffer to 1 μg of working solution with double-distilled water. Return any unused portion to 4°C. 3. Standards: Add 1.0 mL of Universal Diluent for Standards & Samples to the lyophilized standard. Tighten the cap and let stand for 10 minutes to fully dissolve. Gently mix to achieve a concentration of 5000 pg/mL. Perform serial dilutions to 5000 pg/mL, 2500 pg/mL, 1250 pg/mL, 625 pg/mL, 312.5 pg/mL, 156.25 pg/mL, and 78.13 pg/mL. Use the standard diluent (0 pg/mL) as a blank well. Prepare the required amount of standard solution and set aside. It is recommended to add the prepared standard solution to the sample within 15 minutes; do not allow it to sit for extended periods. 4. Biotinylated Antibody Working Solution: Calculate the required volume of biotinylated antibody working solution before use (based on 100 μL/well; add 100-200 μL more). 15 minutes before use, dilute the concentrated biotinylated antibody (1:100) with Biotinylated Antibody Diluent to the working concentration. Use the same day. The dilution principle is to add 1 μL of concentrated biotinylated antibody to 99 μL of biotinylated antibody diluent and mix thoroughly with a pipette. 5. Enzyme Conjugate Working Solution: Before the experiment, calculate the required volume for the experiment (based on 100 μL/well; add 100-200 μL more when preparing). 15 minutes before use, dilute the concentrated HRP enzyme conjugate (1:100) with enzyme conjugate diluent to the working concentration for use that day. The dilution principle is to add 1 μL of concentrated enzyme conjugate to 99 μL of enzyme conjugate diluent and mix thoroughly with a pipette. 6. TMB Substrate - Use a pipette to aspirate the required volume of solution. Do not pour any remaining solution back into the reagent bottle. Notes: 1. Before using the kit, ensure that all components are dissolved and mixed thoroughly. Discard any unused standard after reconstitution. 2. Concentrated biotinylated antibody and enzyme conjugate solutions are small in size and may disperse throughout the tube during transportation. Centrifuge at 1000 × g for 1 minute before use to allow any liquid on the tube walls or cap to settle to the bottom. Mix the solution by carefully pipetting 4-5 times before use. Prepare the standard, biotinylated antibody working solution, and enzyme conjugate working solution according to the required volume and use the corresponding diluents. Do not mix them. 3. Concentrated wash solution may crystallize after removal from the refrigerator. This is normal. Dissolve the crystals completely in a water bath or incubator before preparing the wash solution (do not heat above 40°C). The wash solution should be at room temperature when used. 4. Samples should be added quickly, preferably within 10 minutes each time. To ensure experimental accuracy, it is recommended to use replicates. When pipetting reagents, maintain a consistent order of addition from one well to another. This will ensure the same incubation time for all wells. 5. During the wash process, any wash solution remaining in the reaction wells should be patted dry on absorbent paper. Do not place filter paper directly into the reaction wells to absorb water. Before reading, be sure to remove any residual liquid and fingerprints at the bottom to avoid affecting the microplate reader reading. 6. The color developer TMB should be protected from direct exposure to strong light during storage and use. After adding the substrate, pay attention to the color change in the reaction well. If a gradient is already obvious, stop the reaction early to avoid excessive color that affects the microplate reader reading. 7. The test tubes and reagents used in the experiment are disposable. Reuse is strictly prohibited, otherwise it will affect the experimental results. 8. Please wear a lab coat and latex gloves for proper protection during the experiment, especially when testing blood or other body fluid samples. Please follow the National Biological Laboratory Safety Protection Regulations. 9. Components of the kit from different batches cannot be mixed (except for wash buffer and reaction stop solution). 10. The enzyme label strips in the kit are detachable. Please use them in batches according to experimental needs. III. Operation Procedure 1. Before beginning the experiment, all reagents should be equilibrated to room temperature and all reagents should be prepared in advance. When diluting reagents or samples, mix thoroughly and try to avoid foaming during mixing. If the sample concentration is too high, dilute it with sample diluent to bring the sample within the detection range of the kit. 2. Add 100 μL of the standard or sample to be tested (if the sample needs to be diluted, refer to the sample dilution guidelines for dilution methods). Be careful not to create bubbles. Add the sample to the bottom of the plate well, avoiding contact with the well walls. Gently shake to mix. Cover the plate with a film or film and incubate at 37°C for 80 minutes. To ensure the validity of the experimental results, use a fresh standard solution for each experiment. 3. Discard the liquid in the wells, spin dry, and wash the plate three times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and spin off the liquid in the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 4. Add 100 μL of biotinylated antibody working solution to each well (can be prepared 15 minutes in advance), cover the plate with film, and incubate at 37°C for 50 minutes. 5. Discard the liquid in the wells and wash the plate three times. Wash each well with 200 μL of wash buffer, soaking for 1-2 minutes. Discard the liquid in the plate (or wash the plate using a plate washer). After the final wash, pat the plate dry on absorbent paper. 6. Add 100 μL of enzyme conjugate working solution to each well (can be prepared 15 minutes in advance) and incubate at 37°C for 50 minutes. 7. Discard the liquid in the wells and wash the plate five times. Wash each well with 200 μL of wash buffer, soak for 1-2 minutes, and shake off any remaining liquid from the plate (or wash using a plate washer). After the final wash, pat the plate dry on absorbent paper. 8. Add 90 μL of TMB chromogenic substrate solution to each well and incubate at 37°C in the dark for 20 minutes (shorten or extend the time depending on the color development, but do not exceed 30 minutes). 9. Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow). The stop solution should be added in the same order as the color developer. To ensure accurate results, add the stop solution as soon as possible after the substrate reaction time expires. 10. Immediately measure the optical density (OD) of each well using a microplate reader at a wavelength of 450 nm. The instrument should be preheated and the assay program set before use. Calculation of Results 1. The OD value of the blank well should be subtracted from the OD value of each standard and sample. If replicate wells are used, the average value should be used for calculation. 2. For ease of calculation, although concentration is the independent variable and OD value is the dependent variable, the graphs use the OD value of the standard as the horizontal axis (X-axis) and the concentration of the standard as the vertical axis (Y-axis). To ensure intuitiveness of the experimental results, the graphs present raw data rather than logarithmic values. Due to differences in experimental operating conditions (such as operator, pipetting technique, plate washing technique, and temperature), the OD values of the standard curve will vary. The provided standard curve is for reference only; experimenters should establish their own standard curve. The sample concentration can be calculated from the OD value of the used sample on the standard curve. This value is then multiplied by the dilution factor to obtain the actual sample concentration. It is recommended to use professional curve drawing software, such as curve expert.
Note: This figure is for reference only.
Linearity
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| Species Reactivity | Rabbit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Theory | This kit utilizes a sandwich assay. A specific anti-rabbit MYO antibody is coated in a 96-well microplate. Rabbit MYO standards or samples are added to the wells, allowing the rabbit MYO protein in the standard or sample to bind to the anti-rabbit MYO antibody immobilized on the microplate. Biotinylated anti-rabbit MYO antibody is then added. Unbound biotinylated antibody is washed away, and HRP-conjugated streptavidin is added. After thorough washing again, TMB substrate is added for color development. TMB converts to blue under peroxidase catalysis and to the final yellow under the action of acid. The intensity of the color is positively correlated with the amount of rabbit MYO protein in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the sample concentration is calculated by plotting a standard curve. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Synonym | PVALB; MB；Rabbit MYO(Myoglobin) ELISA Kit | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Detection Type | Can detect recombinant or native rabbit MYO and does not cross-react with other related proteins | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Composition |
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| General Notes |
1. If the entire kit is stored at -20°C, please place the kit at 4°C the night before the experiment. 2. Salt precipitation may occur when the concentrated wash solution is stored at low temperatures. When diluting, warm it in a water bath to help dissolve it. 3. A small amount of water-like substance may be present in the wells of a newly opened ELISA plate. This is normal and will not affect the experimental results. 4. This kit is for laboratory research and development use only and is not intended for use on humans or animals. 5. Reagents should be treated as hazardous substances and should be handled with care and disposed of properly. 6. Always wear gloves, lab coats, and protective glasses to avoid contact between skin and eyes with the stop solution and TMB. If contact occurs, rinse thoroughly with water. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Test Range | 78.13-5000 pg/mL; Sensitivity: 28 pg/mL |
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4.0 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 4
Definitely worth the read!
Format: Kindle
After taking a deep breath and taking in that wicked twist of an ending, I have finally composed myself. My first thought when I started this book was that I love Reyna's character. I was intrigued by her connection with her familiar and the Ruin that is plaguing her land. It came as no surprise that she took her sister's place in an attempt to protect her. When they reach the Air Court everything slows down. This is where it was iffy for me. First of all, I like multiple POV's in books however 7 is a bit much. It starts to interrupt the story line. I felt like I was finally making progress connecting with one character, then it was switched to another person. I felt they all had necessary or pertinent information but not necessarily were they all POV worthy. The only other thing that annoyed me was that Reyna constantly was " trapped." She would rush off without thinking, only to need rescuing. She is brilliant in a fight, but she really doesn't think through anything. Lorcan is amazing. I know he might be on the "bad" list, but his background is so interesting. Eislyn(Reyna's sister) is really so sweet, but calculating. I enjoyed her and Thane's dialogue. The author did an amazing job with the imagery in this book. Everything was so detailed it was easy to fall into the scene. I love unexpected twists and while part of the ending I expected, I wasn't expecting how it took place. All in all, I found it very entertaining and I am very invested in continuing this series.
Favorite quotes:
"The truth may be twisted but never false."
"Who was she if she was not the enemy of the Air Court? What was her purpose of she no longer has that?"
"In a war-torn land, love was always a lie."
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 4, 2020
★★★★★ 5
Great Read!!! Great story!!!
Format: Kindle
The series is long, but Ms. Wolfhart does a fantastic job of weaving this tale while bringing so much to the characters. Surprises and plot twists along the way to keep you intrigued. There is some graphic sex, but is no way the focal point. Grammar was excellent (a rare find with a lot of self publishers) with only a few noted errors. I rarely give 4 stars, let alone 5.
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Reviewed in the United States on April 30, 2021
★★★★★ 3
Sadly, DNF
Format: Kindle
I read this thru KU. I LOVED the synopsis. And then I began reading... and it was a DNF at 68% after picking it up and putting it down several times because I really loved the main female character.
*****SPOILERS*****
Pros:
The world is unique, intriguing and fun. The primary female character is bad-a** but not a b*tech or a mary sue. The primary female has depth. I really want to know what happens to her even tho it's been weeks and I don't remember her name. The villains to the point I read are pretty good -- an ever present threat of mysterious and possibly many culprits.
Cons:
Way, way too many points of view. I stopped counting at 7. It's the prime reason why I don't care about most of the characters or remember their names even when I like them. There's just too many points of view so almost none of the characters have enough book space for the author to properly develop them. This literally killed the book for me. Actually it killed my desire to read. For weeks.
The main male is more villain than hero. He agreed to marry the main female then locks her up & eschews her for her sister, all while bad mouthing her as unfit to rule when he never spent any time with her getting to know her. He is actually unfit to rule as he is blind to the woes of his own kingdom and starts off a peace mission to secure a ceasefire through marriage by murdering an inn full of people in her country for no real reason. Plus, he constantly makes promises he does not keep. And it's gross of him to pine for the sister behind the main female's back.
*****
As much as I really wanted to see what happened to the main female character, it wasn't enough for me to keep trying to slog thru this book. There was a lot of potential here that just fell short.
Hence, 3 stars.
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Reviewed in the United States on June 19, 2021
★★★★★ 5
Fae Courts with High Intrigue
Format: Kindle
This book is one that just builds and builds and then surprises you to no end. You may think you know the villains and then you are jolted in another direction. Princess Reyna is a real gem, strong of character, a fierce fighter, and loyal to her family and kingdom. Just when you think she and Lorcan, well you know, the plot is flipped. Can't wait to see where this goes in book two.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 7, 2023
★★★★★ 4
Slow, sorry but good
Format: Kindle
This was a slow moving book. Lots of character pov chapters, lots of superfluous descriptions and endless courtly appearances stalled this book to start. Once you get into the heart of the story, it takes off. Before you know it the book is done. My favorite character is Reyna. She is so strong. She is true to herself. She gets into a lot of trouble with her headstrong ways but it's entertaining. I have high hopes for Lorcan. He is honorable to a fault. Thane had turned out to be better than I thought but i still don't like him. Eislin is useless. Great plot twists at the end. I'm looking forward too book 2.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 6, 2020
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