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Description
Human NLRP3 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: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend 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. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. 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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| Sensitivity | 0.18 ng/mL | |||||||||||||||||||||||||||||||||
| 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 an NLR Family, Pyrin Domain Containing Protein 3 (NLRP3) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP peroxidase and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of NLR Family, Pyrin Domain Containing Protein 3 (NLRP3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human NLR Family, Pyrin Domain Containing Protein 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | NLR family pyrin domain-containing 3 (NLRP3) (formerly known as NACHT, LRR, and PYD domain-containing protein 3 [NALP3] and cryopyrin) is a protein encoded by the NLRP3 gene located on the long arm of chromosome 1. NLRP3 is primarily expressed in macrophages and, as a component of the inflammasome,436 detects products of damaged cells, such as extracellular ATP and crystallized uric acid. Activated NLRP3, in turn, triggers an immune response. Mutations in the NLRP3 gene have been associated with several organ-specific autoimmune diseases. | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.7 ★★★★★
Based on 868 reviews
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Product Reviews
★★★★★ 3
Ali and the guys
Format: Kindle
The story of a lost art student that needs direction. Decides to nanny for a winery owner and his best friends/employees. Fires and lockdowns happen and relationships start. Lots of spice sometimes a little too much I found myself skimming over the parts. Happy ending
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on January 30, 2026
★★★★★ 5
Artist Inspiration
Format: Kindle
Ali Fairfax is a twenty-seven year old painter. She is going to an interview for a job as a nanny. She wants to go to art college but has no portfolio pieces to submit. For the last year, her mind goes blank the second she picks up a brush. The nanny interview is at Ashford Winery.
Luke Ashford has a son named Charlie who is almost five. Beckett Montgomery is one of Luke’s best friends and he helps Luke run the winery. Hunter Jones is Luke’s other best friend and he is the marketing director for the winery. The men have been running the winery for the last year or so.
Charlie likes Ali and so does Luke, so he hires her. Charlie is into reptiles and he also makes Ali make a grilled cheese sandwich for his snack. Hunter comes in the kitchen to tell them that the winds have shifted and there are wildfires nearby. All the roads in or out of the valley are closed down for now. Ali is stuck at the winery with the men and Charlie.
Interesting dynamic between the three best friends. I loved that being stuck at the winery inspired Ali to start painting again. I enjoyed the story.
I received a free copy of this book via Booksprout and am voluntarily leaving a review.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 30, 2025
★★★★★ 4
So So Good
Format: Kindle
I received this book with the understanding that I could leave a voluntary and honest review.
In this book we meet Ali Firfax, Luke Ashford, Beckett Montgomery, & Hunter Jones. Ali is ready for a new adventure in her life. Though her mother worries about her she is determined to do her own thing. When she gets to the house though she can't believe how attractive all the men who live there are. Luke's first priority is defiantly his son, one look at Alit though has him wondering if he has been to narrow minded. When he two roommates realize who Ali is they will do anything they can to keep her there. When mother nature tends to help them they know they will do anything to keep her. Can they all find a HEA or will mother nature make sure none of them have a future?
Amazing story by this author. I would highly recommend it to anyone.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 13, 2025
★★★★★ 5
Fire 4 ¾ stars
Format: Kindle
Set against the backdrop of California fires, this is a story of Ali finding her forever family. Luke, Becket and Hunter own a winery that is close to the fire. Every day is a wait and see if they are going to lose their home. Ali, an aspiring artist, applied to be the nanny to Luke’s son. The attraction was obvious from the very beginning. Once Ali decided to let go, the steamy scenes were unstoppable. I like that the author showed the readers not just the relationship between her and the guys. The story also showed how the three are close to each other. It’s a good bedtime read. I received an advanced copy and voluntarily chose to review it.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 10, 2025
★★★★★ 5
Great Read
Format: Kindle
The interview was perfect and the way she ran out thinking the worst is so something I would have done. When Luke goes out to get her and asks her to stay is even better. The way Beckett acts is the complete opposite of Luke and Hunter. They all have different personalities but they each pull something different from her. They way they work as a team to save everything was just part of what shows what a future could look like for them. I would love to read more.
I received a free copy of this book via Booksprout and am voluntarily leaving a review.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 8, 2025
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