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Description
Human CYBb 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 preparation and requirements: 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 and
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 preparation and requirements: 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 and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL 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 collect the supernatant for analysis. Cell Lysis Buffer: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended 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 freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. 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 a Cytochrome b-245 Beta Polypeptide (CYBb) 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 Cytochrome b-245 Beta Polypeptide (CYBb) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cytochrome b-245 Beta Polypeptide ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | NADPH oxidase 2 (NOX2), also known as cytochrome b (558) subunit beta or cytochrome b-245 heavy chain, is a protein encoded by the NOX2 gene (also known as the CYBB gene). This protein is a superoxide-generating enzyme that forms reactive oxygen species (ROS). It is the catalytic, membrane-bound subunit of NADPH oxidase. It is inactive until it binds to the membrane-anchored p22phox, forming an isoform known as flavocytochrome b558. Upon activation, the regulatory subunits p67phox, p47phox, p40phox, and a GTPase, typically Rac, are recruited into a complex to form NADPH oxidase on the plasma membrane or phagosomal membrane. | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
Based on 13 reviews
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Product Reviews
★★★★★ 5
Fine Contemporary Poetry--Just Happens to Be Appalachian
Format: Paperback
The poems in Jesse Graves' TENNESSEE LANDSCAPE WITH BLIGHTED PINE express an indebtedness to a way of life that we contemporary Appalachians have watched transform at an accelerated pace over the past few decades, as we see the beloved old ways of our culture adapt to the demands of a society marked with the pervasiveness of media, the incursion of corporate demands, and the poignant recognition that as much as family prepares us to face the world outside our community, the impact of that world can blur the impressions our homes have made on us. Graves' work approaches these themes from various directions, as a son looking to the legacy of his family, as a youth and young man balancing education--both formal and that gleaned from personal experience--and as a family man weighing what he shares and offers in embodying those values.
In this consistently fine volume, it is difficult to select favorites, but there are "River Gods," where an inebriated student and his companion cross the high railway trestle over the Tennessee River in Knoxville, Tennessee, "Deep Corner," where the speaker contemplates how his life has turned out differently than his brother's, "Mother's Milk," where the speaker weighs how much his mother has contributed to his life (including, sweetly, "an ear for slightly off-pitch singing"), and "Digging the Pond," where the speaker and his father silently acknowledge that the son will not preserve all his father's values:
. . . I stood off to the side too often
to learn what he was born knowing.
The doing and the undoing.
I can find in his face what he reads
about the future in the tea-colored water,
his eyes and mine trying to avoid it.
Graves' love for these gifts, those accepted and those only acknowledged, resonates throughout TENNESSEE LANDSCAPE WITH BLIGHTED PINE.
Graves' appreciation for lyric poetry, his talent for finding the expressiveness of everyday language, and his offering scenes with great depth of meaning and feeling make this collection memorable, worthy of high recommendation.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 30, 2011
★★★★★ 5
Extraordinary Journey
Format: Paperback
Jesse Graves conducts the reader on an intimate journey from childhood to manhood. Rooted deep in the rich red clay of East Tennessee, the narrative provides fresh insights about the ties of land and family. "Johnson's Ground" describes an annual homecoming at the family cemetery: "they never let us go, even the ones/Laid under before our births continue to make their claims." The poems express both nostalgia for the past as well as forward-looking hopes for a fresh life in the future. Daughter, Chloe often becomes a bridge from present to past as in "Water Washing Away": "A fair price for the vision of a girl/ who has warped the ancient spell of time,/ who has turned back my eyes." Tennessee Landscape with Blighted Pine is an enchanting read for poet and non-poet alike.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 10, 2013
★★★★★ 1
Go Read Art Smith or Charles Wright
Format: Paperback
This book is clearly the case of someone steeped in a lyric tradition, but, rather than engaging in the self-reflexive structure of the tradition, is interested in describing ad nauseum, his southern experience. While there are moments in the book that tend toward the sublime, it rests largely as self-indulgent in a way antithetical to the form it chooses.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 5, 2013
★★★★★ 5
Dr. G.
Format: Paperback
Jesse Graves (a.k.a. "Dr. G.") is one of my professors at East Tennessee State University. Not only is he a great teacher, he is a very talented poet. I would recommend his work to anyone! Anyone that does not like his work probably just failed his class. :p
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Reviewed in the United States on August 25, 2014
★★★★★ 5
Incredibly Prescient -- Touches On The Major Trends Of Our Times
Format: Hardcover
I was blown away by the fact that the author started writing this book a couple of years ago, before artificial intelligence was in the news, and before the topic and trend of loneliness became so prevalent. The clairvoyance is uncanny and the storyline, while fictional, is totally believable given what's going on in our world today. This is why the novel almost reads like nonfiction, but the plotline carries you forward like a fictional thriller. I audibly gasped, "Holy crap" during the plot twist at the end of the book. Truly a work of art, there is no doubt that this will be adapted for either a movie or a series.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 14, 2024
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