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Description
Human 5-LOX 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 5-Lipoxygenase (5-LOX) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of 5-Lipoxygenase (5-LOX) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human 5-Lipoxygenase ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Arachidonic acid 5-lipoxygenase (ALOX5, 5-lipoxygenase, 5-LOX, or 5-LO) is a non-heme, iron-containing enzyme encoded by the ALOX5 gene. It converts essential fatty acid (EFA) substrates into leukotrienes and a wide range of other bioactive products. The ALOX5 gene occupies 71.9 kilobase pairs (kb) on chromosome 10 and consists of 14 exons and 13 introns, encoding the mature ALOX5 protein of 78 kilodaltons (kD) composed of 673 amino acids. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.8 ★★★★★
Based on 441 reviews
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Product Reviews
★★★★★ 4
Practical Pilgrim Traveling
Format: Paperback
My wife and I earned a compostela walking a portion of the Camino Frances in May of 2004. Since then I've read many books on pilgrimage, including several accounts of other pilgrims' journeys on the same road we traveled. Many are what another reviewer describes: diaries of the interior lives of the author, focusing mainly on their hardships and triumphs, as if to point out how they changed the camino, rather than how they were changed by it. If I felt that this were all to this book, I wouldn't recommend it. Instead, I think this book provides a wonderful balance between soulful reflection and the pragmatism of the all-too-physical journey. Walking the camino does appear to have all the ingredients necessary for earning a 'spiritual experience merit badge', and some seem to walk it just to earn pilgrimage street cred. Even were that Rupp's intention, and I doubt very much that is the case, she's provided a great perspective for potential pilgrims and useful material to aid past walkers. It's true that she does not shy away from describing unpleasantries of the road: dirty accommodations, illness, rude pilgrims, bad food, and bad weather. These are very real likelihoods, and she discusses them very frankly; pilgrims do not float along the road, barely touching the earth, and any idyllic expectations soon come face-to-face with harsh reality. Rupp does not bring up these issues merely to complain, however; the benefit of this book is how she treats these subjects as well as her prayerful introspection as equally engaging points of reflection and provides a useful perspective on integrating even these issues into a larger pilgrimage experience. The subtitle of the book, however, is "Life Lessons from the Camino", and that's the true value of these observations: her effort in showing that much of our day-to-day life is filled with just these sort of experiences and just this sort of potential for reflection, appreciation, and understanding.
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Reviewed in the United States on October 5, 2008
★★★★★ 5
Putting one foot in front of the other
Format: Paperback
I actually bought this book as a gift for a friend who is considering making this pilgrimage. I read it for the first time when it was first published, just because Joyce Rupp is one of my favorite spiritual writers. She has a gift for delving into the spiritual on many levels, from the perspective of a woman, a woman religious, one acquainted with the life and love of God. She writes in an incredibly lucid manner and captures the divine in the midst of life struggles, always prayerfully, with uncommon insight and compassion. In this small and readable volume she tells it like it is.
This book differs somewhat from others I've read in that it is her own lived experience of making this journey across Spain. It's illustrated with photos from that journey and populated and enriched with the varied pilgrims she met along the way.
I recommend it especial for anyone contemplating making this amazing journey, but also for those of us who wish we could.
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Reviewed in the United States on September 9, 2013
★★★★★ 5
Must read before walking the Camino
Format: Kindle
Beautiful, thoughtful account of the many ways walking the Camino can challenge us and help us grow. By far the best of the Camino books I read.
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Reviewed in the United States on October 16, 2025
★★★★★ 3
Not a bad first-person account
Format: Paperback
I had mixed thoughts about this book. It's the author's personal experiences and thoughts about the Camino, but aren't most books about the Camino? I tend to think it's a little too much interior maundering, how every part of the experience affected the writer. Still, what would you expect? I have to call this just an ok read. Most of the reason I liked it at all is because I am intrigued by the Camino and enjoy reading about it.
The writer is a dedicated sister and her companion was a retired priest. I enjoyed the places where she touched on Catholicism, but there wasn't much of that. But there was the part of the book that I found a jarring note, and that was about her take on some fellow Catholics. She and her companion meet a group of three helpful, warm, caring priests and take them to be Jesuits. The priests inform them that that are Opus Dei. As the sister and priest continue walking, they find they are both astounded at the goodness of these men, since Opus Dei is considered to be extremely wealthy, conservative, and have strong ties to traditional Rome. (I thought all Catholics felt they have ties to Rome. I myself talk about the year I "crossed the Tiber.") It is just amazing to this twosome that such nice men could be from wealthy, conservative Opus Dei. I thought this antipathy toward a Catholic group known to do good works told a lot more about the writer than about the well-met priests--maybe more than she intended to let slide about herself. It was the one part of the book that struck a negative note for me.
Other than that, I also wished for more at the end. They finished the Camino and went on to Finisterre. (Huh? What happened to the time spent at the Cathedral at the end? The beauty of the place and the experience of Mass there, and that wonderful incense burner. That whole part was left out.)
I finished the book and consider it just "ok".
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Reviewed in the United States on November 30, 2021
★★★★★ 5
Been on the Camino and love this book
Format: Paperback
I am a Joyce Rupp fan. I'd always dreamt of doing the Camino some day, and when I saw that Joyce had done it, and written a book about it, I quickly bought it and read it.
Her book gave me the courage to buy a plane ticket and go. I'm a hiker and camper. I could tell from reading her book that some of the facets of the hike- some of the albergues, some of the pilgrims, some of the food-- etc etc-- were perhaps harder for her to accept than they would be for me. I thought she gave a really honest appraisal of how things were for her, and was touched by how she eventually resolved some of those contretemps.
I recently was looking at reviews of the book and was surprised to see some of the negative reviews. What I got from reading Joyce's book was an honest look at the Camino from the eyes of a middle-aged woman used to her own personal space, solitude, food, level of cleanliness, etc. One does necessarily give a lot of that up when on the Camino, if you stay in the albergues! They are fabulous places for meeting people from all over the world- but they can make you cringe if you are not used to hearing snoring at night. What I love about this book is the life lessons, her thoughts on what she found there, and what she got out of it in spite of -- and maybe even because of her discomfort.
I recommend this book for mature people thinking of hiking the Camino. In 2011 I accompanied a women's group from my church from Samos to Santiago, and I asked them all to read the book-- they liked it, too.
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Reviewed in the United States on August 22, 2013
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