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Description
Rat DAT 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 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 7 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 directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 dopamine transporter (DAT) 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 dopamine transporter (DAT) content in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Dopamine Transporter ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The dopamine transporter (also known as the dopamine active transporter, DAT, SLC6A3) is a transmembrane protein that pumps the neurotransmitter dopamine out of the synaptic cleft and back into the cell membrane. Within the cell membrane, other transporters sequester dopamine into vesicles for storage and subsequent release. Dopamine reuptake by DAT provides the primary mechanism for dopamine clearance from synapses. The gene encoding the DAT protein is located on chromosome 5 and consists of 15 coding exons, approximately 64 kbp in length. Evidence for a link between DAT and dopamine-related disorders comes from a genetic polymorphism in the DAT gene (DAT1), known as a VNTR, that affects the amount of protein expressed. DAT is an integral membrane protein that removes dopamine from the synaptic cleft and deposits it into the surrounding cell, thereby terminating neurotransmitter 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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★★★★★ 5
exceeded expectations
Format: Paperback
I received this comic book within 10 days of submitting my order. I'm very pleased with the condition of the book - It looks like new! The seller was very accurate with the description. I'll be a repeat customer! It's entertaining reading material for a hard-to-buy-for middle schooler!
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Reviewed in the United States on February 25, 2011
★★★★★ 5
Five Stars
Format: Paperback
Great Simpsons pass time entertainment
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Reviewed in the United States on June 8, 2016
★★★★★ 5
Accessible and insightful guide to project management
Format: Kindle
"Project Management Mastery" describes the science and art of project management in a highly accessible and insightful way. This guide is written in understandable language for beginners and can also be a refresher for seasoned project managers. The guide fully describes the concepts of planning, execution, monitoring and control, and closure.
Each chapter layers new information upon the foundations of the previous chapters by addressing basic concepts and building on details. This book uses project management terminology, but defines it so the concepts are clear, not fuzzy.
Also discussed in depth is the importance of team members' responsibilities and accountability, and the importance of team motivation.
There is a detailed exploration of the differences between various project management methodologies, such as Agile, Waterfall, SCRUM, Kanban and other models.
Also described is the methodology of PRINCE2, used primarily in the UK and Europe, as well as hybrid project management models. There is guidance on which methodology works best for the project being planned.
The guide also addresses the impacts and benefits of how using AI might impact the completion of routine tasks in the project management lifecycle.
Also included is a comprehensive list of references at the end, for those wanting to dive deeper into the subject.
Overall, this is a great guide for new project managers, as well as a solid refresher for those reentering the field, or looking for an updated perspective.
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Reviewed in the United States on June 24, 2023
★★★★★ 5
Mastering Project Management
Format: Kindle, Format: Kindle
What a wonderful book on project management. The author describes what project management is, how to create a project plan, and the different phases that exist. He covers traditional project management and newer versions of it that have evolved, such as Waterfall, Agile, Scrum, and PRINCE2.
The author explains the various types of project management, what are the important tenets of each and which is best for different types of projects. He goes into a lot of detail into each phase, different software you can use, along with visualization tools available like Gantt Charts and Kanban boards.
It is a good comprehensive book that goes in to detail into each area of project management, what you should do to be successful and the various places that project managers typically fail. Finally, he covers final deliverables, project closer and doing a project review and releasing resources. I thought it was a very good book, well done and one that any project manager would love to have as a resource.
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Reviewed in the United States on August 24, 2023
★★★★★ 5
For The Seasoned Project Manager and the Beginner
Format: Kindle, Format: Kindle
It takes a talented writer to write for the expert and the beginner in the same book. But that is how Mr. Hayden writes in this book. It is obvious he is an experienced Project Manager, and he not only gives advice, but gives you an overview of project management in general, and the foundations of it. But then he jumps right into the meat of the matter - and lays out so many ways to start the project, including so many methodologies to use. He includes the SWOT method, that I used with my teams, but there are so many more that I could have used!
I love the way he lays out a plan for you to use - he doesn’t just give you ideas, he lays it out in a way that you can implement it successfully, and more importantly, confidently. That is so important when leading a team!
I highly recommend this book to any level of project manager! And not just those in a project manager position - also for those professionals who are leading a project even though they are in other roles in their company.
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Reviewed in the United States on October 10, 2023
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