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Description
Human FⅧ 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 VIII (FVIII) 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 amount of Coagulation Factor VIII (FVIII) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Coagulation Factor VIII ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Factor VIII (FVIII) is an important blood clotting protein, also known as antihemophilic factor (AHF). Factor VIII is encoded by the F8 gene. Defects in this gene cause hemophilia A, a recessive X-linked blood clotting disorder. Factor VIII is produced in the sinusoidal cells of the liver and the endothelial cells outside the liver, distributed throughout the body. This protein circulates in the bloodstream in an inactive form, bound to another molecule called von Willebrand factor, until an injury occurs that damages blood vessels. | |||||||||||||||||||||||||||||||||
| 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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4.6 ★★★★★
Based on 1003 reviews
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Product Reviews
★★★★★ 5
This would be a terrific book even without the mystery!
Format: Mass Market Paperback
The book has so much literary merit, that it could be placed among the novels. However, the excellent mystery plot also wins it a place among the mysteries.
Haines creates characters who are memorable and vivid and places them in the Mississippi Delta town of Zinnia (I'm kind of reminded of the movie "In the Heat of the Night"). Sarah Booth is living in the ancestral home -- she is an orphan and an only child, the last of the Delaneys -- but is about to lose it, because she is destitute despite her social credentials. Her only company at Dahlia House - the antebellum house -- is the ghost of a slave, who appears in a variety of outfits and "encourages" Sarah to get to work reproducing.
In an attempt to earn some money, Sarah takes on the task of trying to get to the truth of a scandal from 20 years ago in which first a leading citizen and then the leading citizen's wife die in some very questionable accidents. THe two young offspring are whispered to have something to do with it, and Sarah's client wants to find out if Hamilton the Fifth is as bad as rumors have it. Hamilton the Fifth is a romantic interest worthy of Evanovich -- and did I mention the book is often funny?
Sarah is stirring up some dangerous memories and some deaths start to follow.
I really loved this book and can hardly wait to read the next in this series and discover what happens to Sarah.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 19, 2004
★★★★★ 3
Southern Melancholy
Format: Kindle
On the plus side, "Them Bones" delivered on transporting me to the Mississippi delta (how do you stop typing Mississippi? it just begs to go on endlessly, like banana). I was there in the South and in a small country town. Brilliant on setting, which is what I like in a cosy mystery.
The mystery itself took a while to settle in and then, well, I guessed the plot. That meant instead of suspense, I plodded through having my hunches confirmed. But if I'd been charmed, that wouldn't have mattered.
This is not a negative review, but I'm heading towards the big problem I had with the book -- and I think this may be related to the fact that I was so busy and looking to enjoy some simply, happy downtime.
"Them Bones" has humour and a southern feel, but it also struck me as melancholic. And melancholy I did not need. For others (and maybe for me at a different time) that sense of mourning and tangled history might resonate. But it was a heavy load for the plot to carry.
Three stars for a solid read.
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Reviewed in the United States on July 24, 2015
★★★★★ 4
Expect to be hooked
Format: Kindle
Great cozy mystery with characters that become friends in a story that is great 'light" reading for an escape from the usual serious stories and politics on the news. Some laughs, some scares, some regular activities to fill out the characters are in good balance for the genre.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 17, 2019
★★★★★ 5
Not Really a Daddy's Girl Any More..but can sure know how to use it..
Format: Mass Market Paperback
I have at least five of this series that have been in the TBR pile on the bedroom bookshelf..What was I thinking? I absolutely loved this first one for many reasons.One being the rich content that makes up this book, as well as the main protagonist, an Ole Miss Southern Belle, Sarah Booth Delaney, a bit of an outlier when it comes to her former stereotype in the Daddy's Girls(DG's) clique...which is what endears me to her most...never, ever typecast someone as starting out as cut from the same cloth when circumstances aide in their character development..a definite asset in this case. Independent and also in need of money to save the Family home, Dahlia House.and in no hurry to give up her independence for marriage but will consider some stops along the way..when all her former buds have married for economic security rather than love. The dot of on the 'i' is a several generations removed ghost in the form of Jitty..her great, great, grandmother's maid who provides insights and caustic humor throughout. Thrown into these dire circumstances, she latches on to the opportunity to become a Private Investigator. Add a transgender character who happens to work for a local paper in the gossip area, and Sarah Booth is off to the races. Sprinkle in an alleged, physic who is a mainstay of her life long and still successful "DG's, Tinkie, " a murder mystery, a sibling in a psych ward(comfortable of course) a sheriff, and you have all the ingredients of a good ol moss draped mystery. You gotta love it..I know I did.
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Reviewed in the United States on May 6, 2015
★★★★★ 5
Mystery that keeps you guessing
Format: Kindle
This book kept me up reading too many nights because I just couldn't stop reading!! The majority of my reading list consists of murder mysteries. I love trying to figure "who done it?" right along with the main character. My track record for "solving" the case is pretty good but this one had me stumped. I loved that! The author does a great job of giving detail without being overwhelming. The story is of a ex-socialite (Sarah-Booth) who is willing to do anything to save her beloved home. (Think Scarlet O'Hara) In the process of trying to come up with funds, she uncovers a two-decade mystery. She will risk everything, not only to save her home but solve the mystery.
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Reviewed in the United States on September 8, 2014
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