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Description
Human IkBa 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 capture antibody against the inhibitory subunit of NF kappa B alpha (IkBa). 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 amount of inhibitory subunit of NF kappa B alpha (IkBa) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Inhibitory Subunit Of NF Kappa B Alpha ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | IκBα (inhibitor of nuclear factor κB α) is a member of a family of cellular proteins that function to inhibit the NF-κB transcription factor. IκBα inhibits NF-κB by masking its nuclear localization signal (NLS) and maintaining it in an inactive state in the cytoplasm. Furthermore, IκBα prevents the NF-κB transcription factor from binding to DNA, which is required for NF-κB to function properly. The gene encoding IκBα is mutated in some Hodgkin lymphoma cells. This mutation inactivates IκBα, resulting in a chronically active NF-κB state in lymphoma cells, which contributes to the malignant state of these tumor cells. | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.8 ★★★★★
Based on 1675 reviews
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Product Reviews
★★★★★ 5
my new favorite book
Format: Kindle
Ok so I never write reviews but this book was so good I felt the need to write this. Firstly your introduced to Huntyr you see her closed off hard core badass than towards the end you see the most subtle change and growth it’s amazing and the enemies to friends to lovers was just perfect, AND THE TWIST AT THE END GOT ME GOOD! You see one spicy scene the whole book but it doesn’t even MATTER BECAUSE THE BOOK WAS THAT GOOD. I’ve read 85 books in 2023-2024 so far and I’m pround to say this is my all time favorite. I’m so excited to read more of Emily Blackwoods books, this was my first time reading one of hers and I’m glad I did because HOLY!! Well done Emily well done
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Reviewed in the United States on February 23, 2024
★★★★★ 4
Fast paced romantasy you will not want to put down!
Format: Kindle
4.25 stars! I LOVED this book with similar vibes to Hush Hush, Fourth Wing, and The Serpent and the Wings of Night! It was fast paced with easy world building and will keep you turning the pages late into the night because you will not want to put it down!
Huntyr is a fierce bad@ss FMC trained to kill vampyres her entire life. She is sent on a mission to go to the academy and earn her spot into The Golden City. Upon arrival, she is forced to room with the delicious fallen angel, Wolf, who is the only one who knows about her assassin identity. The romance, the plot twists, the secrets revealed, the battles, and the tantalizing training scenes had me hooked! And that ending…. I’m holding my breath in need to know hell!
Read if you love:
🪽 Fae, Vampyres, Fallen Angels
🪽 Academy setting with magical trials
🪽 Forced proximity and slow burn
🪽 Rivals to lovers
🪽 Hidden identities and secrets
🪽 Tend your wounds
“𝘖𝘧 𝘤𝘰𝘶𝘳𝘴𝘦 𝘐 𝘸𝘢𝘴 𝘸𝘢𝘵𝘤𝘩𝘪𝘯𝘨 𝘺𝘰𝘶. 𝘐 𝘤𝘰𝘶𝘭𝘥𝘯’𝘵 𝘭𝘰𝘰𝘬 𝘢𝘸𝘢𝘺 𝘧𝘰𝘳 𝘢 𝘮𝘰𝘮𝘦𝘯𝘵, 𝘦𝘷𝘦𝘯 𝘪𝘧 𝘐 𝘵𝘳𝘪𝘦𝘥.”
“𝘐𝘧 𝘺𝘰𝘶 𝘸𝘢𝘯𝘵 𝘮𝘦 𝘰𝘯 𝘮𝘺 𝘬𝘯𝘦𝘦𝘴, 𝘏𝘶𝘯𝘵𝘳𝘦𝘴𝘴, 𝘢𝘭𝘭 𝘺𝘰𝘶 𝘩𝘢𝘷𝘦 𝘵𝘰 𝘥𝘰 𝘪𝘴 𝘢𝘴𝘬.”
“𝘠𝘰𝘶 𝘥𝘰 𝘯𝘰𝘵 𝘬𝘯𝘰𝘸 𝘵𝘩𝘦 𝘷𝘪𝘰𝘭𝘦𝘯𝘤𝘦 𝘵𝘩𝘢𝘵 𝘳𝘶𝘯𝘴 𝘵𝘩𝘳𝘰𝘶𝘨𝘩 𝘮𝘺 𝘷𝘦𝘪𝘯𝘴, 𝘣𝘦𝘨𝘨𝘪𝘯𝘨 𝘮𝘦 𝘵𝘰 𝘰𝘣𝘭𝘪𝘵𝘦𝘳𝘢𝘵𝘦 𝘢𝘯𝘺𝘰𝘯𝘦 𝘸𝘩𝘰 𝘭𝘢𝘺𝘴 𝘢 𝘩𝘢𝘯𝘥 𝘰𝘯 𝘺𝘰𝘶.”
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 12, 2024
★★★★★ 5
Excellent Rivals to Lovers!!
Format: Kindle
The tension and banter between Huntyr and Wold was delectable. I absolutely love the fallen angel and all of his flaws. Huntyr is amazing too being a badass FMC with some major trauma. The world building was great and I enjoyed the training aspect of the story. The writing was immersive and was in the story the whole time. The ending had quite a twist that I hadn’t anticipated and made my jaw DROP. Excellent job! I also loved the narration. Laura is one of my fave narrators!
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Reviewed in the United States on August 15, 2025
★★★★★ 5
Fallen Angels, fae, vampires, oh my!
Format: Kindle
Rating: 4.5 | Spice: 2 (but a good slow-burn)
•
Main Characters: Huntyr and Wolf
•
I couldn’t wait to read this book; there was so much hype about it! And there was no doubt why. I fell in love with the characters and the plot itself. This book is mainly plot driven more than friction driven but it’s easy to follow along with. The characters are fun, easily understood. The main setting is at an academy where both the main characters are going through trials and building strength for the final test, The Transcendent. There are fantastic side characters as well. I loved the camaraderie between Huntyr and her friends. But we don’t like Lanson. 😆
We do have some plot twists that come into play throughout the book. Secrets and betrayal to be seen. I did adore Wolf and Huntyr’s relationship. It was a classic slow burn trope. They didn’t hit it off fast, but in time their feelings grew. I loved their banter, so sexy. Wolf is your next book boyfriend; Huntyr is your next vampire assassin independent bad-a*s female. Themes include loyalty, trust, self-discovery, a true slow burn romance. Side note: book ends on a angsty cliffhanger!
•
Emily, thank you for writing this awesome novel and I cannot wait to devour Book 2, Blood So Brutal! 😍
•
Happy reading, my lovelies! xo
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Reviewed in the United States on January 21, 2024
★★★★★ 4
“My heart bows to you and you only, Huntress.”
Format: Kindle
3.5 🌟
This book popped up in my KU recommended reading suggestions and the synopsis sounded like what I was in the mood for. I'm so glad I took a chance on it. I went into this knowing absolutely nothing about it and ended up really liking it. I love when this happens.
The main characters are likeable and I easily found myself rooting for them. There is a mystery element to each of their backstories that I enjoyed watching unfold and can't wait to get more of. Wolf, in particular, has me fixated. Love him.
I found this to be an entertaining, addictive read with a plot that moves along at a good pace. It reads so easily I found myself very reluctant to put it down. Lots of twists and turns and the angst is there. A good set up for the next book to come, for sure.
My issues with this book....the dialogue feels a bit juvenile at times and there is a repetitive over use of a particular word phrasing that I found myself giving the ole eye-roll to. There are, without a doubt, some pretty cliche moments that gave me a bit of the cringe. I think this could've certainly 100% benefited from more depth regarding the world building. Perhaps the world building was sacrificed to keep the pacing quick? Just a guess. Also, the lack of consistency of character for the FMC was really evident and so she feels quite illogical at times.
Overall, this was a fun and enjoyable read that hit the spot well enough for me. That ending certainly has me impatiently pining for book 2!
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Reviewed in the United States on June 18, 2024