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Description
Rat MuRF1 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 utilizes 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 Muscle Ring-Finger Protein-1 (MuRF1). 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 Muscle Ring-Finger Protein-1 (MuRF1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Muscle Ring-Finger Protein-1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | The E3 ubiquitin-protein ligase TRIM63, also known as "MuRF1," is encoded by the TRIM63 gene. This gene encodes a member of the RING zinc finger protein family, found in striated muscle and iris. The gene product is localized to the Z-line and M-line lattices of myofibers, where the N-terminal and C-terminal regions of titin, respectively, bind to the myofiber. In vitro binding studies have shown that this protein also directly binds to titin near the region containing kinase activity. Another member of this protein family binds to microtubules. Because these family members can form heterodimers, this suggests that these proteins may serve as a link between myosin kinase and microtubule-dependent signaling pathways in muscle. MuRF1 is the most commonly used name in the literature, standing for "muscle RING Finger 1." Structurally, there are two closely related MuRFs, MuRF2 and MuRF3. These also have the TRIM code. MuRF2 is TRIM55, and MuRF3 is TRIM54. | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.4 ★★★★★
Based on 21 reviews
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Product Reviews
★★★★★ 3
So close to being good
I really wanted to like this book. It had all the makings of a strong, interesting story, but the plot got so convoluted that it was hard to follow. Also, for a "badass" FMC, Milla was amazingly inept. The writing got really stilted at times too. I will probably still read the next one anyway, because I'm curious to see where the plot goes.
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Reviewed in the United States on May 3, 2024
★★★★★ 5
FASCINATING world, great characters and swoony slow burn!
House of Bane and Blood by Alexis Menard
-Order and Chaos Book 1-
4.5/5⭐️
1/3🌶️*
2/3🦋
•My Thoughts•
What a RIDE! This world was absolutely fascinating with its 1920s mafia vibes meets magic. The characters and enemies to lovers storyline hooked me from the beginning - add in a marriage of convenience and a dash of mystery and I ate👏🏻it👏🏻up👏🏻. Milla’s growth was so good and I was cheering for her as she became more confident! I also loved Niko and his back story and resilience. These are imperfect characters you find easy to love, root for and I became enamored with their slow but steadfast (if reluctant) support of each other. I did find the government structure a little confusing but didn’t want to slow down enough stop and figure it out (if anyone reads it an wants to make a chart, please share it with me). This book was violent but also mesmerizing and full of hope for a better future. I loved the big family dynamics and the never ending plot twists that kept me guessing. This is a completed duology and with that ending, you BET I’m immediately reading book 2.
*I rated this a 1/3🌶️ because there were only a few scenes with on page intimacy. However, I would like to note that the spice involves kink: breath play.
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Reviewed in the United States on June 10, 2024
★★★★★ 4
Top tier romantasy
I really enjoyed this book! It's everything a romantasy should be, really. The interactions between the characters was good, the world was interesting, and I liked the magic system. Slow burn was a 10/10! I really love Nico. He's so intriguing and I hope we get more of his POV in the next book.
Reasons for taking off a star:
1. I didn't think the world building was set up well. I got the full picture by the end, but the author writes as if we should already understand and it was a bit confusing for quite a while.
2. Milla's characterization was inconsistent. She was wishy washy with her goals and didn't give off the bad ass vibes I think the author was aiming for. I would have also liked more showing and not telling for her background.
3. (This is me being a bit ridiculous) I didn't know if I was supposed to pronounce it as "Mih-luh" or "Mee-luh." I think the former sounds silly but my brain didn't want to read it as the latter 😅
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Reviewed in the United States on November 7, 2023
★★★★★ 5
Give me a little bit’a that Nicolai Roman Attano ANY DAY. 🥵
⭐️ 5
🌶 3
🚂 Peaky Blinders Vibes
🚂 Enemies to Lovers
🚂 Sexy + Sassy Banter
🚂 Marriage for Business
🚂 Unique Magic System
🚂 Dual POV
I absolutely LOVED this book. The mafia/peaky blinders vibes — the lil’ steam punk feel mixed with magic and mayhem… add in the witty banter from Milla and Nico? ALEXIS, YOU DONE JUST ROCKED MY WORLD, FRIEND.
What I Enjoyed:
Nicolai. Roman. Attano.: Literally. This is all I’ve got to say to you, just read the book.
The Banter: When I tell you the banter between Milla and Nico is iconic — I mean it. Scroll down for some of my favorites, but my my my, my core was HEATING and I was wriggling in my seat on the airplane, my friends. OOOOOMPH.
Steampunk/ Peaky Blinders Vibes: I read this whole book with a feeling of mist on the ground, the flickering street lights, sound of a train horn, flapping of coat tails, and smell of cigarillo smoke. It was dark, broody, beautiful, and chilling. Add in a little wetness from morning dew and the scene has been set.
Plot Twists & Turns: The amount of times my eyes bugged out of my head with “OMG NO WAY”, “WHAAAT”, and “BOOIIIIIII–” should almost be illegal. I didn’t see the twist at the end coming, I loved it! Brilliant! I feel like House of Bane and Blood didn’t even allow me the opportunity to guess what would happen next, because I just had no idea where Alexis would take it.
Attano Family: I’m a sucker for a big family with many cousins, personalities, and a spicy Nonna. Throw in a kitchen scene or two and you’ve got me hooked. I loved the dynamic between the Attano family and how fast they came to bat for Milla. I throughly enjoyed getting to know the people behind Nicolai, and how they all support him and each other.
What I Didn’t Like:
The Minimal Insight on the Marchese Family: We aren’t really given much insight into the Marchese Family and Milla’s brothers. I understand this gives an ominous feel to the brothers — but I really wish we could have explored Giles and Milla’s relationship just a little bit more. I wish I understood more about the Twins than what was given. Although, I do understand the distance and the coldness, I personally just wanted a little more.
Over All Thoughts
The way I straight flew through this book — UGH. Adding Nico to my list of book boyfriends, and Alexis to my list of instant buy Authors. The way House of Bane and Blood is written is beautiful, it does not feel immature or overwhelming, there’s enough to keep you riveted and more and more unfolds with every chapter. Milla and Nico are a force to be reckoned with separately, but together? Indestructible! I cannot WAIT to see what happens in City of Mirth and Malice.
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Reviewed in the United States on July 9, 2024
★★★★★ 4
A Promising Start with a Complex Magic System
“House of Bane and Blood” delivers an engaging enemies-to-lovers romance wrapped in an arranged marriage trope, which was one of the strongest aspects of the book. The dynamic between Nico and Camilla was compelling, making their evolving relationship one of the highlights of the story. The plot itself was interesting and had a lot of potential.
However, the complexity of the magic system was a drawback. The abundance of difficult names and terms made it challenging to fully grasp the world-building, leaving some aspects feeling more confusing than immersive. At times, it was hard to keep track of what was happening and who was who.
Despite this, the book holds promise as the first installment of the series. With an intriguing setup and strong character chemistry, I’m excited to see where Book 2 takes the story. Hopefully, it will continue to build on its strengths while offering more clarity on its magical elements.
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Reviewed in the United States on March 17, 2025