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Description
Rat ERFE 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 them by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with ice-cold PBS and resuspend them 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, or store at -20°C or -80°C, but avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge at 1000×g for 20 minutes, and remove the supernatant for analysis. Pre-Assay 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 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: Add 500 μL of universal diluent to each of seven EP tubes. Pipette 500 μL of the 10 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5 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 erythroferrone (ERFE) capture antibodies. 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 erythroferrone (ERFE) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Erythroferrone ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| 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 homogenates, cell lysates, cell culture supernatants and other biological fluids |
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4.5 ★★★★★
Based on 22 reviews
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Product Reviews
★★★★★ 4
Banter, a bit of angst, found family, and a hot, tattooed bad boy who’s really a totally softie
Format: Kindle
This was a fun, easy read. It has all the things I love in a sports romance...banter, a bit of angst, found family, and a hot, tattooed bad boy who’s really a totally softie.
Knox is kind of a mess at the start. He’s super talented, but has a reputation problem after getting kicked off his motocross team. He’s desperate for a second chance, which leads him to Avery, a former Olympic gymnast recovering from an injury. She’s trying to get her confidence back, and somehow these two end up helping each other.
They can't stand each other at the beginning. He says ALL. THE. WRONG. THINGS. Evenutally, their friendship builds into something more. It felt natural and sweet. I liked that they both had their own stuff going on. Avery’s inner struggle was really relatable, and Knox’s bond with his brothers, especially with Flynn, added a ton of heart to the story.
The beginning was little slow, and the ending felt a little rushed. Overall, though? I had a great time with this book.
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Reviewed in the United States on July 27, 2025
★★★★★ 3
New Type of Sports Romance!
Format: Kindle, Format: Kindle
Instagram: @notyourkindlehistory
Burnout by Rebecca Jenshak
Rating: 3.5
Spice: 3.5
Tropes:
- Gymnast x motocross rider
- Training together
- Tattooed hero
- College romance
- Ton of Banter
My Review
Okay… I’m not even sure how to start this review so I’m just going to explain my 3.5 rating. I want to begin saying, there really wasn’t anything that made this book bad. I have read previous books by Rebecca Jenshak, and I LOVED them, rated them 4 or 5 stars, but for some reason, this book took me about 35-40% of the way through to finally get into it and enjoy it. Once I got past that point, I absolutely loved it. I’m not sure if it was a mood I was in, or just that it was a completely different type of sports romance, but something just didn’t grab my attention soon enough. I loved Avery and Knox, two complete opposite kind of people, which made for a ton of banter. I loved that we were able to get inside of the head of Avery, an Olympic gymnast, who was recovering from an injury, and see what that process of the road to recovery and competing again at 100% looked like from a mental stand point. Knox was this character that you got immediate bad boy, I don’t do relationships and just hook up vibes from, but slowly throughout the book you can see Avery and Knox going back and forth and breaking each-others walls down. They began hanging out more and doing more things together, but didn’t want to define what they had because it worked for them and what they needed at the time. This book had different aspects of a relationship that you don’t see in other romance books, such as long distance. I thought the end of this book was left more open ended than closed, and I felt it just kind of ended, or the epilogue wasn’t really a cherry on top, it was more so just the sprinkles. BUT, overall, I would recommend this book to read to others; the spice in this book was AMAZING (there may or may not be a spicy motorcycle scene hehe), the banter was great, we really got a feel and understanding of each character too. I wonder if I read it again, or read other books in the universe to get some background on this story, maybe my mind will change on my rating; but for now it’s a solid 3.5 for me.
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Reviewed in the United States on June 13, 2024
★★★★★ 5
Good book
Format: Kindle
This is my first book by this author and I loved it so much. Such a cute story. I absolutely loved reading Knox and Avery's story. Knox is a motocross racer and Avery is a gymnast. Knox lost his spot with his team for not being a team player. He is bummed out because racing has been all he really wanted to do. Knox lost his mom when he was a teenager and he took on a pretty big responsibility helping take care of his brothers. He has an older brother who went to college and then two younger ones that also ended up going to college and then there's the youngest one that's a senior in high school. Knox is a great big brother but really wants to race so his friend Colter, who does freestyle tricks on bikes, talks him into trying to do trick riding too.
Avery hurt her knee a bit ago which she did therapy for but is really aware of her knee now and just doesn't have the confidence like she used too. Avery goes out with her friend to a party to get out of her head and there she sees Knox. He's on his bike but he almost got her and from there it starts there relationship of having snarky comments to each other. Avery helped Knox' friend Colter do some gymnastic workouts to get his strength and learn some tricks for his trick riding. Colter recommends Knox work with her too. Knox takes him up on this because Knox needs this to show people in motocross racing he can be a team player.
Knox and Avery end up getting a long better and you can feel the chemistry between the two but both don't want a relationship for different reasons. Things heat up between the two of them. Lots more happen in this book and you will want to read to find out what that is. Will Avery and Knox be able to get over their fears and be together? Have to read to find out. Thank you Rebecca Jenshak for this book.
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Reviewed in the United States on April 28, 2024
★★★★★ 4
Solid start to a new series! Love the Holland brothers
Format: Kindle
There is one thing I've learned when I read Rebecca books: I either love them and am obsessed (looking at you "Tutoring the Player") or I really like them, but feel like there's something missing. And this book fell in the latter.
Ok, so, I did really enjoy this book. I was so excited to get to know the younger Hollands after meeting Hendrick in "Tempting the Player." I was so intrigued by their dynamics, so when I saw those Holland brothers were getting a series, I was SOLD. And I'm not gonna lie, I really enjoyed grumpy and closed-off Knox Holland. The relationship he has with his brothers is special, but especially the one he has with his youngest brother, Flynn. And that, alone, made me fall in love with him. But watching as Avery wore him down, without even trying, was the best. And how he didn't even realize it was happening? Even better.
Avery was awesome! I LOVED her! She's had a rough go after an injury at the end of her freshman year at Valley U, and it's really rocked her confidence as she works to come back. She was funny and the way she fit in with the Holland brothers was perfect... especially the way she cared for Flynn. I loved the banter and way she goes back and forth with Brogan too. That guy is hilarious. She just...fits, and they all can see it.
The only thing that kept this from a 5 star for me was that it just felt like it...ended. We have the conflict, we get back together, but then, it's a super quick last chapter, no epilogue, and it's over. I really wanted to see at least a little more of them as an official couple. I do love how you could see how close she and Flynn are in the last chapter. And how even though the others have moved on to other things (Archer and Brogan the NFL, Henny and Jane are married), they're still such a tight family unit.
There is a super short but sweet epilogue you can get at the end of the book if you sign up for Rebecca's newsletter (which, duh, I already get because I do love her books!). Overall, I really enjoyed this book, but I have to say, the level of excitement I have for Archer and Flynn, but Archer especially, is next level!
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Reviewed in the United States on June 22, 2025
★★★★★ 5
I loved it so much!!!
Format: Kindle
I've had this one on my TBR lis forever and been meaning to get to it. Considering I picked up the ebook last year not long after it came out. I can't believe I let this one sit on my kindle for as long as I did. I ended up starting and finishing in just one afternoon absolutely hooked on Avery and Knox. They has this connection that just works even if they are to stubborn to see what's happening. I loved the whole bad buy meets the good girl in this sweet steamy enemies to lovers romance.
The author does a beautiful job creating a captivating story with characters you can't help falling in love with. This is my first book by the author and I'm a fan as I enjoyed every moment of Avery and Knox's story. I loved that we get to know a little bit of Knox's brothers as the series is about them and can't wait to devour there stories too. Highly recommend the characters are engaging and the story is perfectly balanced with just the right amount of romance. This one made me want to look up the riding that Knox did off season and it's impressive. I loved it!!! One of my favorite books of 2025.
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Reviewed in the United States on May 5, 2025