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Description
Human NLRC5 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 competitive enzyme-linked immunosorbent assay (ELISA). Samples, standards, biotin-labeled antibodies, and HRP enzyme conjugates are added sequentially to microwells pre-coated with universal species NLR family CARD domain containing 5 (NLRC5) antigens. After incubation and washing, the sample is developed with the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. The depth of the color is negatively correlated with the universal species NLR family CARD domain containing 5 (NLRC5) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human NLR family CARD domain containing 5 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The NLRC5 protein (NLRC5) is encoded by the NLRC5 gene. It is also known as NOD27, NOD4, and CLR16.1. It is an intracellular protein that plays a role in the immune system. It is a pattern recognition receptor and may be involved in innate immunity to viruses by regulating interferon activity. In lymphocytes, NLRC5 localizes to the nucleus and drives MHCI gene expression by occupying the H-2D and H-2K gene promoters. | |||||||||||||||||||||||||||||||||
| 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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4.1 ★★★★★
Based on 15 reviews
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Product Reviews
★★★★★ 2
Has Potential, Poorly Executed
Format: Kindle
I really really wanted to enjoy this book and was looking forward to reading it. However there were just one too many convoluted plot points, sentences just did not flow nicely at times, and I was left with a lot of confusion on my end. I think it has the bones of being a great story but seems like it was rushed and I had to push myself to finish the last of the book and was left with an unsatisfying ending. It’s definitely different from other omegaverse novels I’ve read but not for good reasons. I really think it could be great but needs some serious tweaking and editing before I’d read it again.
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Reviewed in the United States on February 3, 2025
★★★★★ 5
Secrets, Seduction, and Rockstars!
Format: Kindle
Rockstars, secrets, and off-the-charts chemistry? Sign me up!
Cinder Blaze takes us on a rollercoaster of passion and peril with Knot Their Omega. Blair Vesper, a secret Omega masquerading as an Alpha, strikes a risky deal to tour with Blooming Salvation, a band teetering on the edge of chaos. Enter Icarus Morrigan, the enigmatic manager, and his three complicated and irresistibly sexy rockstars: wild Kenji, icy Kaiser, and fiery Nathaniel.
This book delivers steamy Omegaverse drama, sizzling slow-burn romance, and just the right dash of angst. The tension between Blair and the band crackles like electricity onstage, while the societal stakes add depth to the spicy dynamics.
Short, sharp, and oh-so-sinful Knot Their Omega will have you hooked from the first note!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 9, 2024
★★★★★ 5
Good start to a series
Format: Kindle
I delayed reading the series for reasons I don’t remember. But my TBR list is huge so I thought I’d take a shot of this and I was pleasantly surprised. I didn’t think the blurb about it was anything special. But it was a very good book. It took some interesting twists and turns. I am so glad the second book is already out. Because I would not have waited patiently. Very slow burn but good storyline. 🔥🔥/5
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Reviewed in the United States on January 3, 2025
★★★★★ 4
A good read
Format: Kindle
Multiple points of view. 3 Alpha men and an Omega male. She is a Beta in training for a new program placing betas in Alpha/Omega packs. Mila is only doing the program for the money to take care of her dad. She wasn't expecting to fall for a pack but when she sees this packs Omega she is done for. There is just something about him. His Alphas are good looking as well. Too bad she is hiding a secret and their government is acting shady. I liked it and can't wait to see where their story goes.
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Reviewed in the United States on November 14, 2023
★★★★★ 3
Slightly repetitive but I did love some things
Format: Kindle
I love this type of story. And omegaverse is one of my all time favorite genres. But there are a few things that pulled me out of my enjoyment while I was reading. It was repetitive at times as well as struggled with telling not showing. So we didn’t always feel like we were experiencing things with the main character. There were also some plot holes but they may still be answered in part 2.
Now this isn’t to be said I didn’t enjoy parts of the story. I loved the almost instant love between Mila and Oliver. And how he started changing around her.
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Reviewed in the United States on February 15, 2024