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Description
Human 1433PR 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 capture antibody against 14-3-3 protein (1433PR). 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 14-3-3 protein (1433PR) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human 14-3-3 protein ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. They bind to a diverse array of signaling proteins, including kinases, phosphatases, and transmembrane receptors. Over 200 signaling proteins have been identified as 14-3-3 ligands. Elevated levels of 14-3-3 proteins in cerebrospinal fluid may be a sign of Creutzfeldt-Jakob disease. | |||||||||||||||||||||||||||||||||
| 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, and other biological fluids |
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4.1 ★★★★★
Based on 161 reviews
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Product Reviews
★★★★★ 4
A mystery and a healing journey for 3 people
Format: Kindle
⭐⭐⭐⭐
🌶️🌶️🌶️
Touch her and 💀
🍑🍆🍑 MFM/Why choose
🤓Military MMCs
🥺Damaged hero
🤯BIG mystery to solve
🐶Lovable fur bestie
😈Forced proximity
My Thoughts 💭
I definitely feel the book is contemporary but there are some dark sides to it just because of their past and what they went through within the military as far as the guys.
I can say that I do love how Aiden and Miles are such good friends and how they stuck together and have helped each other.
Aiden is very goofy and free willing but has a lot of guilt and almost in a way self-conscious.
As were Miles gives off a dark and broody daddy vibe and Aspen ends up being the one to just unravel him.
I do love Aspen work and how she overcomes what she's been through and how she sees her growth and her self-worth and decides to put herself first.
This is a very much meat cute moment just because of the way they meet but then you have the backstory of like Miles and all of that...
I feel that the underlying plot of the mystery aspect really keeps you entangled into everyone else as of the side characters so I'm excited to see more of the series.
Spice is that a three just because I feel like there was so much more to their relationships and their interactions that the spice wasn't the forefront of it which was good because they needed to grow all three together and you need to see how they interacted separately and there was a lot of trauma healing in this book so it's understandable why the spice isn't the forefront.
I would say that if you're looking for something that is going to be sweet but yet pull at your heartstrings this will be the book.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 16, 2025
★★★★★ 3
Left with Questions
Format: Kindle
I'm hoping some things will be wrapped up in book 2 but with the epilogue for this book being a year later I'm currently just confused. Otherwise a very good read.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 24, 2025
★★★★★ 5
Love, Love, Loved!!
Format: Kindle
This book was a wild and fun ride. It has suspense, it has found family and most importantly it had so much true love and friendship. I fell in love with Miles and Aiden instantly. The love and affection between these two friends was so profound and deep you just instantly wanted them to find their HEA. I absolutely loved how they supported one another through all their hurt and trauma. They have this unbreakable bond that is truthful and real. It’s simply beautiful how they hold up one another no matter what. When they meet Aspen not only does she fit into the town so perfectly, but she is able to appreciate each man for who they are instantly. She’s a great FMC in that she’s so kind, genuine and willing to trust again even when she’s been hurt by people in her past.
This book had a journey filled with so much love and healing that my heart cried several times. Each character was able to not only break through walls, but help one another grow into better versions of themselves. The love that flourishes when all three come together was electric and passionate. The spice was steamy and the romance was swoon worthy. The mystery throughout the book is one that isn’t completely solved in this story so I’m looking forward to the next book in the series and of course seeing more of Miles, Aiden and Aspen. 5 stars!
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Reviewed in the United States on June 20, 2025
★★★★★ 5
Miles, Aiden, and Aspen
Format: Kindle
Man this was a great start to a series. The author got you hooked one the book from chapter one. We are still don't have all the answers but they will come in due time. I absolutely enjoyed this story. Miles seriously has my heart. He was just such a lovable guy. He served his country and was injured doing his job. He carries around a lot of trauma and emotional damage from that time. His childhood best friend Aiden feels guilt over what happened to Miles. He blames himself even though it was not his fault. Due to his trauma they form an unlikely pairing. They need each other and to share the person they are with due to Miles's trauma. They met Aspen and form and instant connection. Little does Aspen know that she has helped Miles during some of the worst of his times with her beautiful pictures. She is running away from a relationship and a job that literally used her and tossed her away. She forms an instant connection to both guys and Miles's dog Jubie. She teaches them both to not only forgive their past but also love themselves more. She is everything they need in their life. Mean while there is a killer/stalker running around causing havoc. Will we find out who the bad guy is? I guess we will have to keep reading the series to find out. I really enjoyed this book and I think the author did a great job keeping your interest. I cannot wait to see what else she has instore. She had great world building and character development.
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Reviewed in the United States on May 20, 2025
★★★★★ 4
Rediscovering herself and finding the unexpected
Format: Kindle
First we have Aspen a young woman who lost herself to her ex and what he wanted. She ran to Alaska to re-find her love of photography and the outdoors. While she did find this she also found a broody and overprotective former Navy Seal who still had a lot of healing to do, his super sweet over thinking best friend and their lovable doggo.
I absolutely love the different plot lines going on in this story. It leaves mystery and a sense of foreboding around every turn. Of course this doesn't answer all the question we have for Anchor Bay because this is only book one but I absolutely love the community they have.
Aspen has an immediate connection with the two friends. She has an easy and open demeanor that puts even the broody guy at ease enough to engage. Aspen is sweet, open, and genuinely cares about the people and animals she comes in contact with.
I can't wait to see what is next in Anchor Bay.
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Reviewed in the United States on May 17, 2025
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