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Description
Rat Rac1 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 Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) 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 Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Ras Related C3 Botulinum Toxin Substrate 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ras-related C3 botulinum toxin substrate 1, also known as Rac1, is a protein encoded by the RAC1 gene. This gene can produce multiple alternatively spliced forms of the Rac1 protein, which appear to have distinct functions. It is a small signaling G protein (more specifically, a GTPase) and a member of the Rac subfamily of the Rho family of GTPases. Members of this superfamily appear to regulate a variety of cellular events, including control of GLUT4 translocation to glucose uptake, cell growth, cytoskeletal reorganization, antimicrobial cytotoxicity, and protein kinase activation. It is a pleiotropic regulator of many cellular processes, including the cell cycle, cell adhesion, motility, and epithelial differentiation. It is ubiquitously expressed in tissues and drives cell motility by forming lamellae. It is abundantly expressed in insulin-sensitive tissues, such as adipose tissue and skeletal muscle. It is also required for glucose uptake in skeletal muscle, which is activated by exercise. | |||||||||||||||||||||||||||||||||
| 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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4.5 ★★★★★
Based on 30 reviews
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Product Reviews
★★★★★ 5
wow!
Format: Kindle
What an amazing story that tells about history from the perspective of a kid named Ben that was pretty much forced (but was thankful for the work) to work with coal in the Tower of London, to find out Princess Elizabeth was arrested and sent to the same tower. The plot thickens as he helps her escape. He was met with many challenges, but his best friend Tim helped him always, what a faithful friend! I absolutely love and adore this story because it’s based on history and real life facts, these things actually happened! But when you read it, the author has written it so well it almost sounds like a fantasy! I can’t wait to read more, it’s such a great book, I give it 5 stars but really it deserves 10! Highly recommend!
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Reviewed in the United States on October 20, 2021
★★★★★ 5
Don't let this great middle grade book escape you.
Format: Paperback
I am an Anglophile and love the history of England. This book was an incredible read and fed my addiction fully with Renaissance England inspired history and fiction featuring Ben, a 15 year of commoner and an accidental hero who finds himself a prison let working the Tower’s dreadful jail. This spry has short chapters that will appeal to children with a short attention span, and the storyline like the other installment feature children using real life problem solving skills as they help others. This is an overall great read and highly recommended.
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Reviewed in the United States on August 18, 2021
★★★★★ 5
Suspenseful
Format: Kindle
Ben a 15-year-old found himself chained to a wall in a dungeon. His job was to carry coal around the towners to rich prisoners. Princess Elizabeth was thrown into the traitor's tower by her evil sister Queen Mary. He had to try and help save her, now he is a prisoner. The adventure starts, will Ben get out? How will he ever get out? will the princess be saved? Every page is filled with suspense.
I loved the history lesson at the end of the book, filled with so much information.
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Reviewed in the United States on August 16, 2021
★★★★★ 3
A Tudor Tale of Derring-Do for Children
Format: Kindle
Ben Forrester sees a young woman being dragged into the Tower of London and despite the good advice of his friend, Tim and it being a seemingly impossible task, he sets out to rescue her. On the way, there are messages written in invisible ink, a princess dressed as a coal boy - a coal boy dressed as a princess - dungeons with rats and a lot of adventure.
This is an exciting and well-written tale for children, set in the reign of Mary Tudor in the violent and cruel days of the sixteenth century. It has a lot in it to acquaint a reader with life and society at the time and gives a (highly distorted) sketch of the political events of the time.
My one big complaint as a historian is the biased perspective of this book which reinforces many popular myths and misconceptions about both Mary and Elizabeth. As this book might be the first (and for some children maybe the only) experience they have of the period it seems to me tragic that it does so.
To read it you would think Elizabeth was perfect and Mary pure evil. We are told of Mary's burnings but not why they happened or of Elizabeth's subsequent persecutions and none of it is placed in context.
This is a black and white version of history which is horribly inaccurate because it is so, and misleads another generation to despise the woman who was England's first true Queen and who overcame so many challenges including those of politics, religion and rabid misogyny to rule in her own right.
I love the irony that we find out in the historical notes at the back that the story of Ben's escape was based on the real-life escape of a man imprisoned there by Elizabeth on account of his beliefs...
So whilst I can applaud this as a good, immersive, book to help a child learn about the social history of the time, I would hesitate to recommend it because of the way it reinforces the ignorant bias of popular history over the truth.
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Reviewed in the United States on October 4, 2021
★★★★★ 5
Great for kids
Format: Paperback
Trying to encourage my outdoor loving grandson to read
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Reviewed in the United States on June 2, 2026