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Description
Human NF-KBp50 ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 nuclear transcription factor p50 (NF-KBp50) 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 nuclear transcription factor p50 (NF-KBp50) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Nuclear transcription factor p50ELISA kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Nuclear transcription factor p50 (NF-KBp50), also known as NF-κB1, is encoded by the NFKB1 gene. NF-κB is a transcription factor activated by various intracellular and extracellular stimuli, such as cytokines, oxidant free radicals, ultraviolet radiation, and bacterial or viral products. This gene encodes a 105 kDa protein that is co-translationally processed by the 26S proteosome to produce a 50 kDa protein. This protein is a specific transcriptional inhibitor of Rel proteins. The 50 kDa protein is the DNA-binding subunit of the NF-κB (NF-κB) protein complex. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.0 ★★★★★
Based on 22 reviews
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Product Reviews
★★★★★ 3
Interesting, but not empirically validated, and written explicitly for straight people (why?!)
Format: Kindle
This one’s the 500-pound gorilla of the healthy-couple self-help genre, with thirty years in print, scores of Oprah appearances by the authors, thousands of therapists practicing the Imago framework.
The main thing that I need to tell you about this updated edition, from the year 2019, is that it does not mention or acknowledge the existence of queer or same-sex couples even one time. In fact, it really doubles down on the husband and wife stuff, takes gender roles pretty seriously, which is not a thing I realized we did anymore, except as a joke. Anyway, talking only to straight people is not too relevant to my interests, and while the conservative Christian viewpoint is pretty subtle in the advertising, just be aware of that PROBABLY NOT ACCIDENTAL omission going in, queer friends.
(This is made even more impressive by the fact that the heart of the Imago approach is based on childhood trauma, about the ways in which our parents caused us to suppress or hide or ignore parts of ourselves in order to be accepted and loved. Just let that sink in, because I’ve been reading this book for the past week and I am still reeling from the irony that people who have been thinking for the past three decades about the ways that parents cause trauma to kids by failing to accept parts of them have missed the VERY OBVIOUS AND RELEVANT EXAMPLE of queer folks.)
That aside, for me the book was a mixed-bag of some interesting and provocative ideas (I’d put these in the category of poetic notions, or interesting narrative ideas), alongside some really kooky 20th century neo-Freudian stuff (rooted in part in Freud’s theory of repetition compulsion.) I’m no fan of Freudian approaches, and after I started this book, I decided to see how empirically-validated this Imago approach is: based on what I found, there seems to be basically no evidence that this therapy is effective.
Am I glad I read it? I think so?? It was thought-provoking, with the pretty significant grain of salt that there’s not much evidence to support that Imago therapy is much better than doing nothing at all. Also, I feel a little gross giving my money to relationship experts who (in 2019!!)) won’t even acknowledge the existence of queer people, even when doing so would obviously strengthen their core arguments and provide better examples than some of the meek ones they give.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 18, 2021
★★★★★ 5
Great book!
Format: Paperback
I read this once through on my own and picked up a lot of great wisdom. Now my spouse are reading it together so we can answer the questions together. I'm excited for thought provoking discussions on raising our future child.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 4, 2020
★★★★★ 5
Now you know (some of) what you don't know
Format: Kindle
As I'm ready to embark on parenthood, I truly appreciated the insights offered by these parents. The learning has given me more confidence and preparation in managing expectations, handling difficult situations and being the ideal parent.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 12, 2019
★★★★★ 5
As an expectant parent, I found the information in ...
Format: Paperback
As an expectant parent, I found the information in the book to be helpful. While I know I will not come across all the topics immediately, I know I can keep it as a reference in my journey of parenting.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on January 14, 2017
★★★★★ 5
Thinking of having kids?
Format: Kindle
This book opens up more that just the kids question. It's the working in a team with your spouse that will build a strong house! It's not easy, but you you need to work at clear matters before u enter parenthood. This book does that.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 26, 2016