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Description
Human SLC7A11 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 utilizes 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 cystine/glutamate transporter (SLC7A11) 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 cystine/glutamate transporter (SLC7A11) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cystine/glutamate transporter ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Solute carrier family 7 member 11, also known as SLC7A11, is encoded by the SLC7A11 gene. This gene encodes a sodium-independent, chloride-dependent cystine-glutamate antiporter, termed system Xc- or xCT. It regulates synaptic activity and non-vesicular glutamate release by stimulating extrasynaptic receptors. This gene is highly expressed in astrocytes and couples the uptake of one cystine molecule with the release of one glutamate molecule. Dimeric cystine is taken up by glial cells, while monomeric cystine is taken up by neurons. Xc- expression has been detected throughout the brain, with higher expression found in the basolateral amygdala, retina, and prefrontal cortex. Inhibition of system Xc- has been found to alter numerous behaviors, suggesting a key role in excitatory signaling. | |||||||||||||||||||||||||||||||||
| 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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4.9 ★★★★★
Based on 2360 reviews
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Product Reviews
★★★★★ 5
Well done
Format: Hardcover
Beautiful book. Big, beautiful illustrations. The reading is easy though some of the concepts may be a little difficult for younger ones to grasp. Though this just leaves a great opportunity for further discussion on all the different reasons for crying. Great teaching tool. The illustrations do a wonderful job depicting these deeper concepts. Readers may find themselves lingering a little bit on each page, after the reading is done, absorbing the meaning of the words and pictures. Very well done.
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Reviewed in the United States on May 17, 2020
★★★★★ 5
Beautiful book, sweet message
Format: Hardcover
Go ahead and buy it for all the kids you know
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 11, 2021
★★★★★ 5
Sensitively written and beautifully illustrated
Format: Hardcover
Why Do We Cry? is an sweetly illustrated and sensitively written book for young readers. Originally published in Spanish in 2018, this English translation released 7th April 2020 from Kids Can Press is 34 pages and available in hardcover and ebook formats.
The book takes the form of a dialogue between a mother and son about why people cry and why it's good to examine and experience negative emotions so we can process them and what function tears (and crying) actually serve physiologically and emotionally. The text by Fran Pintadera (weblink in Spanish) is supported and enhanced beautifully by the nuanced illustrations by Ana Sender.
This would make a superlative reading circle, library group, or classroom read for youngsters. There's a short discussion resource at the back of the book which includes some science based information on tears and the physiological functions they perform.
Learning to process strong emotion in a positive and constructive manner is -vital- for development. I appreciated that the dialogue was sensitively handled and that the author chose to make the child male was a nice touch. I think that this could be a very valuable book *especially* given our current pandemic world situation.
Five stars. Beautiful little book with universal relevance.
Disclosure: I received an ARC at no cost from the author/publisher for review purposes.
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Reviewed in the United States on April 19, 2020
★★★★★ 5
A thoughtful book to normalize tears
Format: Hardcover
I'm a child life specialist I have heard many parents and providers tell kids (and teens) to "stop crying" or "there's nothing to cry about" or some other variation of that hurtful phrase. I now work with children who have a parent with a serious illness and those experiencing grief. Why Do We Cry? is an amazing resource that is relevant to the majority of situations experienced by kids in my practice. It is beautifully illustrated with images that reflect the emotion in the written content. This book embraces the many reasons people cry without overstepping a boundary by trying to explain or justify the feelings. I love the subtlety of normalizing a variety of feelings and the gentle truth that sometimes we cry without knowing why. I highly recommend this book!
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Reviewed in the United States on December 10, 2022
★★★★★ 5
5 Stars!
Format: Hardcover
Mario asks his mother, “Why do we cry?” and she goes on to tell him all the different reasons that we do cry. Some of the reasons are a bit metaphorical, and might be harder for younger children to understand, but the illustrations help in those cases. (The book is aimed for 3-7 year olds, but I think the story is more fitting for ages 5 and up). The illustrations correspond well with the words on the page.
The story lets children know that it is ultimately ok to cry. I feel that the book showcasing a boy asking about crying is wonderful as some boys are told NOT to cry.
A few added pluses:
There is also a lesson that scientifically explains about tears so children can understand some of the varying reasons that tears exist. There is also a small interactive section with activities dealing with tears that will help children express their imagination.
Many thanks to Kids Can Press for granting me an e-arc to read that I chose to review. A+ on this children’s picture book!
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Reviewed in the United States on April 7, 2020
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