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Description
Rat F5 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 handling and requirements: Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000g for 15 minutes at 2 8C within 30 minutes of collection. Remove the supernatant for testing or store at
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 handling and requirements: Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge at 1000×g for 15 minutes at 2-8°C within 30 minutes of collection. Remove the supernatant for testing or store at -20°C or -80°C, but avoid repeated freezing and thawing. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare a gradient standard working solution: Add 1 mL of universal diluent to the lyophilized standard. Let stand for 15 minutes to completely dissolve, then gently mix (concentration 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 seven 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 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 a Coagulation Factor V (F5) 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 Coagulation Factor V (F5) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Coagulation Factor V ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Coagulation factor V (FV) is a protein of the coagulation system, rarely referred to as a pro-accelerator or vulnerability factor. Unlike most other coagulation factors, it lacks enzymatic activity and instead functions as a cofactor. Defects can lead to a bleeding tendency, while some mutations (most notably factor V Leiden) predispose to thrombosis. The factor V gene is located on chromosome 1 (1q24). It is genomically associated with the multicopper oxidase family and is homologous to factor VIII. The gene spans 70 kb and consists of 25 exons, producing a protein with a relative molecular mass of approximately 330 kDa. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | plasma |
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4.5 ★★★★★
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Product Reviews
★★★★★ 5
A Book I Wish I Had Back Then
Format: Paperback
This is EXACTLY the sort of book I wish I had available to me when I was ready to leave home. I was filled with all sorts of braggadocio and I knew everything I needed to get out the door..............and then was introduced to the 'school of hard knocks'
From discovering how to budget and manage your money, to learning how to cook and eat healthy food, to figuring out how to start a career, this book has something to teach even the most well rounded youngster. These aren't in-depth discussions about every aspect of how to live your life, but these ARE a great starting point and with the knowledge provided here, and a little bit of common sense, you can survive early adulthood and move towards a more fulfilling life.
Life is a series of learned events, usually set it motion through observing the people we respect, but not everyone can be everywhere at once and this book can and does fill in a bit of this and a bit of that, often inspiring a conversation that will provide more opportunities to share life lessons and avoid some of the bumps and bruises life has to offer.
As a grandparent watching his oldest grandsons get ready to leave the nest, I found this book to be a helpful way to share my experiences in a way that goes well beyond "because I said so" and reaches into the 'how' and the 'why', even explaining the reasoning, all without seeming to meddle in their affairs.
Unlike childhood where you can stomp your feet, declare 'that's not fair' and receive an immediate 'do-over', real life doesn't offer that and it's best to be prepared for what life is going to bring you. This book does that.
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Reviewed in the United States on April 6, 2025
★★★★★ 5
life skills
Format: Kindle
This is a great book full of life skills. the opening up managing your money and budgeting. The book is full of 100 everyday life skills that we need in order to function. ones that we might not have been taught growing up. the most significant chapter for me was on the money and I had to deal with stress and anxiety.
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Reviewed in the United States on June 7, 2022
★★★★★ 4
Some information may be new...
Format: Paperback
My son overall liked the book, but did say quite a few things listed he already knew.
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Reviewed in the United States on January 11, 2023
★★★★★ 5
Great boob!
Format: Paperback
I love this book I think everyone who is 18+ should get there hands on this it gives a lot of good tips and ones that you never thought about until you read the book. Overall I would totally recommend this book I would give it more than 5 stars if I could.
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Reviewed in the United States on January 16, 2023
★★★★★ 5
Overview of like skills for teens
Format: Paperback
Bought for grandchild's 16th birthday.Over view of life skills and steps to becoming a young adult
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Reviewed in the United States on February 17, 2024