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Description
Human Kindlin-2 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: 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: 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. 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. 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. 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. 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. 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 kindlin-2 capture antibody. 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 kindlin-2 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Kindlin-2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Fermitin family homolog 2 (FERMT2), also known as pleckstrin homology domain-containing family C member 1 (PLEKHC1) or kindlin-2, is a protein encoded by the FERMT2 gene. Kindlin-2 was the first kindlin protein discovered in 1994. It was discovered during a screening for epidermal growth factor (EGF)-induced mRNAs and was originally named mitogen-induced gene 2 (Mig-2) protein. | |||||||||||||||||||||||||||||||||
| 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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4.8 ★★★★★
Based on 15 reviews
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Product Reviews
★★★★★ 4
The series as a whole so far 5/25
Format: Kindle
I read reviews before going into this book and I don't agree with one of the more harsh ones on the main trigger she had. It is stated clearly in the forward and it wasn't as blase as it was made out to be. It definitely is touched on more and hasn't just been brushed off as the series goes
I definitely would recommend reading it. It's a good series just be for-warned
I like the series as a whole. The characters are awesome I adore the fmc shes cute and adorable but also a badass. Though there are a bunch of holes for her that I feel like just got left out.
The guys are interesting and shout out to yall for not making Gage a dragon. I'm tired of the broody ones who don't wanna talk aboit what they are being Dragons. Ki is my favorite
You can definitely tell if is written by 2 different people though because the phrasing just doesn't match up and wouldn't be something people that age says. And it flip flops between them.
I feel like there's substance without substance. We are 4 books in and we don't really know much back story on literally anyone more than right under surface deep.
There are definitely favorite MMCs which is kind of disappointing since some get shoved to the wayside. Specifically both of the best friends. They're basically useless and it's made obvious as the books go on. As well as all the men are ungodly self deprecating.
I enjoy the plot line for the most part like I said I enjoy the series its different and refreshing.
I do feel like the series is being dragged out though unfortunately. And the latest cliff hanger was just meh. So hopefully the next book is the last one.
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Reviewed in the United States on May 8, 2025
★★★★★ 5
Wow!
Format: Kindle
This book was awesome! Seraphina and her family have moved to a new town. Her family is a bit... odd. She grew up learning how to protect herself from people who might hurt her. Bloodshed is a daily occurrence with her brothers and parents during their practice sessions, and it’s all fun and games unless you need to hide a body.
Sera’s family is very close, and she’s been homeschooled most of her life. But in this new town she is going to start regular school as a senior at the local high school. Unfortunately, things at her school aren’t all they seem to be. Or perhaps more than they seem to be. Sera has her own demons to deal with, and she’s terrified her new friends will learn about her weird family and other issues and drop her like a rock. It turns out they have their own secrets as well.
This story ends on a bit of a cliffhanger and I can’t wait to read the next one! This book is well written and well edited. The heroine is spunky and has a great heart and wicked sense of humor.
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Reviewed in the United States on November 12, 2021
★★★★★ 5
Great New Fantasy Series!
Format: Hardcover
I came across The Road of Bones by Demi Winters as I was scanning NetGalley and when I read the summary I was intrigued. I'm always interested in a good Fantasy but when you throw Vikings into the mix, I'm on board. Now, I did have this book on my TBR shelf, but I'm going to tell you, thankfully when I cracked this book open I happened to be on vacation because I couldn't put it down and I've been telling everyone I know who will listen that they need to read it. Not only because it is good but because I NEED someone to talk to about it.
Winters starts this book out with a bang and pulled me in from the start...Silla Nordvig is our fmc and she is on the run. We find out very quickly that she and her father have been moving from village to village but we don't truly know why and unfortunately as they are headed on their way out of their most recent village, Silla's father is killed and Silla is on her own trying to survive. What she doesn't understand is why the queen of Iseldur has sent warriors to bring her back to Sunnavik and based on her father's last words to her, Silla is determined to travel the Road of Bones and get to Kopa and the safety of a shield house.
After barely surviving her first stretch of travel, Silla overhears a group discuss their travels and she sneaks into their supply wagon. What she didn't count on was that the wagon belonged to the notorious Bloodaxe Crew. To make it to Kopa, she has to win over Axe Eyes, the crew leader, while not falling for the Wolf's handsome face. As she finds herself getting closer to the whole crew (and slowly winning them over), the queen's hunter is on her tail and is determined to bring her back to Sunnavik no matter what it takes.
Like I said, I couldn't put this book down. There is non-stop action in this book. Whether it is the danger of traveling the Road of Bones and coming across the various monsters there or trying to escape the queen's assassin, the Bloodaxe Crew and Silla are always facing some sort of danger along their travels.
As for the characters, I love a book that has me feeling all the things for all the characters and this book did this for me. First, there is Silla. I won't say a lot about her backstory as I don't want to ruin anything here but she is put through the ringer here. That said, she doesn't ever give up and I love that about her. Don't get me wrong, she has her moments where she questions everything and doubts herself just like anyone who has gone through what she is going through would but she always tends to sit in it for a moment and then pull herself back together. And despite all this, Axe Eyes still refers to her as "Sunshine" which compared to him is probably fair.
Wolf/Jonas - He values family and loyalty so that is a plus but I have a lot of feelings here and I can't say anything for fear that I will give anything away and sway someone's thoughts. That said, I had high hopes for him and I will talk about him more in my review for book 2.
Axe Eyes/Rey - As grumpy as he likes to come across, he truly is a protector and while he likes to pretend he doesn't like Silla around, he also doesn't truly want her gone and when things go wrong, he is the one who tends to come through for everyone. Again I won't say a lot here as he has some secrets that are revealed and feed into the next book.
Rest of the Bloodaxe Crew - The rest of the crew are a great supporting cast and help to move the story forward.
Ultimately, this book was one that did a great job of combining the character's stories with the overall plot and there is an underlying story here. It's not just about Silla trying to get to Kopa. The queen of Iseldur is up to something and while we only get snippets of what the queen wants Silla for through her assassin/hunter, there is definitely something bigger at play. Admittedly, it wasn't a surprise to me as to what Silla's background was but I also couldn't help from feeling all the feelings for Silla as she starts to realize what she is up against and what that means for her. Also, it's a harsh world that these characters live in and Winters did a fantastic job of putting it down on paper and getting me absorbed into it.
If you're looking for an adult fantasy to pick up, consider grabbing this one. It has a great story, strong writing, solid world-building, and well-developed characters. Plus...then we can talk about all the things. The next book, Kingdom of Claw, comes out early in 2025 so you won't have too long to wait to see what happens with these characters. I'll definitely be picking up the next books in the series to see what happens for these characters as this book/series has me in a chokehold.
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Reviewed in the United States on November 5, 2024
★★★★★ 4
4.5 ⭐️ for this epic Viking Romantasy!
Format: Kindle
I slept on this book for far too long! I understand the hype now, and it is most definitely well deserved.
While I thoroughly enjoyed this book, it did start a bit slow for me. I was a bit confused by the magic system as we never really got a good picture of how it works. But alas, it did pick up mid way through and I devoured the remainder of the book.
There are so many things to love about this book - the found family, the characters, the perilously danger filled journey and the plot twists. There were enough Easter eggs that I was easily able to figure out the twists before they were revealed. The romance also comes with a bit of a twist and I knew it wasn’t right. It felt a little forced and was slightly uncomfortable to witness knowing he wasn’t the right love interest. By the end of the book, I was giddy with glee and look forward to seeing if what I hope will transpire comes to light in the next book.
As this is a Viking setting, it comes with a lot of violence and gore, so make sure you take that to heart if you pick up this book.
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Reviewed in the United States on December 29, 2025
★★★★★ 5
Call it a Masterpiece
It is such a pleasure to revisit The Road of Bones. To travel its road with a fresh perspective and fall in love all over again-- as if it's the first time. For it is a revelation.
The world-building that daunted me the first go around is now a warm embrace, it is an exhilarating charm. I readily hold onto its every detail, I listen up for every piece of information, collecting ravenously. I fall in deeper love with our heroine Silla and the enigmatic Bloodaxe crew.
This book should be considered a classic. I hope it is, I hope one day when people list modern classics, this gorgeous story (and its glorious sequel that is soon to be re-released under Delacorte Press!), will be firmly at the top of all mentions. That’s where I’d put them.
The Road of Bones opened my eyes to a brand new world, and I feel right at home.
A home led by whom has become one of my all-time favorite heroines, Silla. Oh dear Silla, how I adore you. My favorite part of her is her strength, but it’s not the type ever so commonly seen in fantasy. It’s not one of brawn and physical power, it’s one of brain, wit, and so much heart. I can’t express how much I NEED heroines like her. Soft, gentle, vulnerable, and oh so wholly relatable. I was glad she wasn’t a warrior, but that doesn’t mean she’s not a fighter. That doesn't mean her own unique strength isn’t as valuable as those who can wield a heavy blade or physically stand in the way of the villains. Her strength is her courage, her kindness, her wit, her will to live and never bring harm to others. She will buckle, she will fall, but she always tries her very best to get back up. And she feels so much, she wears her heart on her sleeve, and is vulnerable, is emotional, and is so utterly human.
I look at Silla’s journey in awe, I look at what’s ahead in wonder, and I look at that girl and think: she is magnificent. She defies the archetype of the common fantasy heroine and that makes her all the more unique and relatable.
Something this book achieves for me that no other book had until now, is make me love a multi-POV book! I was nervous, y’all! Could I handle more than single or dual POV? As it turns out: Heck yeah I can! In fact, I highly anticipated each POV presented as the narrative unfurled. But I played favorites, I can’t lie, I did, I did!
Silla, my girl, my number one. But her love interest— *the* love interest— slowly but surely creeps in the most tantalizing way. I don’t want to spoil this POV, but my story’s end, trust me, you’ll know. And he is such an incredible part in this story, his scenes at the end had my heart racing and I genuinely mean that literally. My heart was pounding in my ears, both times I read this. I think every time I reread the ending all my emotions will be pouring out like it’s the first time. I was just so immersed, my attention so deeply captured that it like connected into each of emotions in an explosion of fireworks. It’s so rare to find a book that pulls something so powerful out of you.
But The Road of Bones does just that.
Brava, Demi Winters, brava! To watch this journey from an indie published work to now a traditionally published book, allowing for greater audiences, has been absolutely magic.
If you love fantasy, romance, intelligent plots, excellent pacing, and a dynamic cast of characters you will come to know deeply and feel so many colliding emotions for— then pick The Road of Bones as soon as you can!
You won’t regret it— that is my promise to you! And just you wait, oh my goodness, just you wait for the new edition of Kingdom of Claw, to me— it’s the greatest sequel of all time, and I am so excited to read its new release. Let’s goooo!
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Reviewed in the United States on November 5, 2024