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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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4.7 ★★★★★
Based on 6 reviews
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Product Reviews
★★★★★ 5
Well holy moly!
Format: Kindle
Holy moly that story definitely had me hooked!
Kieran was definitely a character that I immediately adored. She has obviously had to deal with a butt load of too high expectations from her father (who is 🤬)..
She has been helpless when it came to finding out her affinity with so many years of no answers & perceived failure. When Ronan, gives her a small inkling of hope that she be a Beast Tamer, she jumps into it. But again she doesn’t seem to fit.
But when she runs into Gabe again, she ends up helping him & rethinking her life in Alfemir. And when the opportunity comes she decides leaving is the best option. But when Ronan demands he comes with her & then the random appearance of Bastian, she is feeling very overwhelmed.
After she chooses to fall with them, they land on earth exactly where Gabe is located only to run into Steele who is a person that I was finding hard to like.
After training, she eventually finds out she does have an affinity & it’s one that no longer exist & is incredibly powerful but also has a prophecy attached to it.
I feel incredibly bad for her on she learns about the prophecy & Steele’s connection with it too.
But that ending, with the battle scene & Niz….yeah definitely was not expecting that but hello I am all here for it too!
Absolutely amazing start of a series & I for sure will be waiting for the next one to come out.
Amazing job!
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Reviewed in the United States on September 26, 2024
★★★★★ 5
So good!
Format: Kindle
Wings of Stars was a fantastic first book to a new series. It grabbed me in its clutches within the first chapter and kept ahold of me.
This new world of M Sinclair and RL Caulder’s is all about angels and mythical creatures. It’s full of drama from all points. From Kieran’s awful home life to keeping the stars alive and everything in between.
Kieran is our FMC and despite everything she keeps her spirit and defiance.
Keeping to their norm, there is a group of guys for our FMC but they definitely aren’t established yet, and not fully cemented yet into being a group rather than her choosing between them.
Ronan is so sweet and gives off Daddy vibes
Gabe is also sweet and wants the best of everything for Kieran and will defend her against anyone
Bastian is slightly unhinged but I loved him immediately
Steele is a jerk… just as you find redeeming qualities, he screws up again… but yet I still want him to redeem himself.
There’s another one… but if I gave his name I would be spoiling a plot twist so you will just have to find out yourself… but you’ll love him as much as I do.
This is a slow burn but the little bit of spice we do get is hot, especially with the dirty talk.
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Reviewed in the United States on February 19, 2024
★★★★★ 5
Angels and the Fallen — Breathtaking Start
Format: Kindle
This book was EVERYTHING I expected it to be and more. Leave to R.L. Caulder and M. Sinclair to give us yet another amazing book!
Kieran and her guys hooked me from the very beginning and did not let go. Found family is one of my favorite tropes and it was done so well in this book. Couple that along with an FMC trying to find her place in the world and I was practically drooling over this book. The world building, the conflict, the wyverin sidekick — it all was done so well that it felt fresh and real.
Each love interest felt real and unique in their own ways. I felt connected to each one and like they were truly different people. I love that so much in a book and these two authors never miss with their love interests.
Multi POV, Reverse harem, who did this to you, and a magical world highlighting the angels and the fallen. This book has everything and it does it so well.
Big thank you to R.L. Caulder and M. Sinclair for the arc copy! I cannot wait for book 2!
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Reviewed in the United States on February 20, 2024
★★★★★ 4
A new take 🤔
Format: Kindle
I’m not going to go into what this book is about — by now you e already read the synopsis and have an idea — but I will give a couple of insights.
Definitely a different take on angels and their fall; I like it. This is an interesting start to a new series - YES, this does end on a cliffhanger so be prepared for that. I’m eager for the next book (and hopefully the last in this series — sometimes the author(s) will switch it up depending on how the story flows for them) in this series.
I’m hoping that a certain male character redeems himself because he’s a dick that will make the meaning of mixed signals jealous lol.
You ever watch that old movie — I think it’s call Mommy Dearest? — yea, let’s switch that up to daddy. That man would make a perfect husband to mommy dearest.
I recommend this book because while these authors are well known for their cliffhangers, they are also well known for putting out good stories.
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Reviewed in the United States on February 19, 2024
★★★★★ 3
Liked it
Format: Kindle
I liked the worlds and the characters. The prophesy is confusing in that I would think, since all worlds ore on a path to destruction, the angels would want their world to be saved. I am sure there is more evil that will be revealed as the series proceeds. I dislike a prolonged “poor little me” trope and the main character has moved past that, thankfully, and began to show a fierce attitude as the novel progressed. The last portion of the novel had some great action. It’s worth reading, just be patient with a slow start.
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Reviewed in the United States on February 14, 2024