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Description
Rat cdc25a 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 Cell Division Cycle Protein 25 (cdc25a). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of Cell Division Cycle Protein 25 (cdc25a) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Cell Division Cycle Protein 25 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Cyclin 25a (cdc25a), also known as M-phase-induced phosphatase 1 and dual-specificity phosphatase Cdc25A, is a protein encoded by the cyclin 25 homolog A (CDC25A) gene. CDC25A is a member of the CDC25 family of dual-specificity phosphatases. CDC25A is specifically degraded in response to DNA damage, leading to cell cycle arrest. It is considered an oncogene because it cooperates with oncogenic RAS to transform rodent fibroblasts and is overexpressed in tumors of various tissues, including breast and head and neck tumors. It is a target of the E2F family of transcription factors. Therefore, its overexpression is a common consequence of dysregulation of the p53-p21-Cdk axis during carcinogenesis. | |||||||||||||||||||||||||||||||||
| 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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4.2 ★★★★★
Based on 1042 reviews
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Product Reviews
★★★★★ 5
Epic cliffhanger
Format: Kindle
All I can say about this book is WOW!
Absolutely loved the characters in this book. Raven is such a bad ass chick.
Brax
Creed
Zane
Eldon
They’re all such good characters my favorite is Brax even with his brooding personality at first.
This book is a PNR with such great chemistry between the characters right away. I love the snap backs between them all. There is a bit of mystery thrown into the story as well. We don’t get to know what everyone’s gifts are right away an I like that a bit it helps us get to know the characters first in my opinion.
Eldon is the one who falls first then the others Raven is sorta stand off ish mostly because she has no idea as to what life is like at Silvercrest. She was brought up in Shadowmoor an we find out why towards the end. Her brother is a pain he’s so full of himself I have a feeling he an the dad have a sinister plan for some reason.
The connection between the guys an Raven is a strong one. Eldon has his visions that he this has to do with Raven but you get a glance at them as well.
I don’t like Finn an Genie you’ll see when you read.
I love how Raven instantly makes friends with Leila regardless of what others think. I hope we get more of Raven and her familiar the cliffhanger was a mind blown thing. I’m excited to see where that goes I’m not typically a PNR person but this story just drew me in an I’m glad it’s so good it’s not a dark romance as I tend to go towards but I love it it’s a breath of freshness an I think a lot of you would definitely enjoy this series remember it’s a reverse harem so you don’t have to pick a favorite guy 😏
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Reviewed in the United States on October 2, 2023
★★★★★ 4
I’m excited! this was Fun!
Format: Kindle
I really liked this! This is a new author for me. I was looking for something with Fantasy and magic and I think I really scored a good one! A lot of this book leaves you guessing and trying to fill in the blanks with Raven about what’s being kept from her and why’s she’s had to live the life she’s had. I was really confused by her family, it’s definitely disfunctional. Her brother wants to kill her, her father is a major a22 hat, her mom looks like she needs major help to escape from her husband. They really threw me off. I knew it was going to be something big and the clues are dead give away. Even though it leads you to outcome it’s still a fun read, lots of banter and HOTTNESS going on! I loved her guys, they came on really strong and it just works. I’m looking forward to the next book. The only reason I didn’t give it 5 stars is how predictable it is any sometimes raven was a bit annoying. It was enjoyable but I did put it down a few times and come back later. It has my interest.
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Reviewed in the United States on February 15, 2024
★★★★★ 5
Pink Raven…
Format: Kindle
I am convinced that KC Kean can write anything and I will absolutely love it. From dark romance to fantasy she’s taken me on a ride of ups, downs, twists, turns and some pretty brutal cliffhangers. This book didn’t disappoint in bringing it all and all I can say is give me more.
Raven’s journey to Silvercrest Academy brought with it some rough patches in her life. She spent 14 years in a place of darkness fighting everyday to survive. Only for her father to bring her back to a deceiving light.
When her father returns for her, she’s surprised with his decision to send her to Silvercrest Academy especially since she is a void. She has no magic or atleast that’s what she thought. When she arrives she sees her brother for the first time in 14 years and he hasn’t changed, he’s still the angry spiteful little boy that she knew back then.
She’s placed in a house with four of hottest guys she’s ever seen. Eldon, Brax, Creed and Zane are first years and her new roommates. Their relationships grow over time and we get to see some of their magic over trials that are meant to break them.
I really enjoyed this first book. Raven is tough on the outside and for good reason. We sense a different side to her once she feels a connection with the one’s she cares about.
We do get left on a slightly soft cliffhanger and I can’t wait for the next book to see what happens next.
Fantasy setting, steamy bits and an unknown dark force that has started to show its intentions. Yes! Give me more!
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Reviewed in the United States on October 4, 2023
★★★★★ 5
falling in love with this book 🖤
Format: Kindle
This book captured me from the very beginning. Raven is an absolute baddie. She grows up in one of the roughest places and then gets tossed into an academy that is made to break the students who attend.
When she arrives she finds herself in a house with four roommates Zane, Eldon, Creed and Brax by accident.
The guys are protective from the very beginning and I love that. Raven doesn’t take any crap from anyone and seeing her have that support that she hasn’t really ever had is really sweet🖤
Then there’s her brother Sebastian 🙄 who for some reason can’t stand Raven even though he hasn’t been around her since she was 6. He is an absolute twatwaffle.
Then the gauntlet is brought out to play and for some reason it’s being done differently than it is usually done. Everyone has to participate in it and Raven is without magic to help her escape the gauntlet with her life. Getting to see each of the guys magic and the affinities that they possess is so freaking exciting. And then Raven meeting Ari 😍😍😍 Though I do feel like that last outpost visit to shadowmoore was a set up to get her to use her magic that they think she has. Ugh this book kept me pulled in to the very last word.
7/5⭐️’s
2.5/5🌶️’s
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Reviewed in the United States on October 4, 2023
★★★★★ 3
Nicole
Format: Kindle
This book is more of a 2.75! First off, the beginning is just awful. We are dripped into a scene that we know barely about the people and then face forward like 12 years or something. There is little mention of how life was hard and miserable in shadow Moore but boom the dad is there and she hates him. Mom, where's mom. Oh, she's back at home. What?!?!! You're going to some academy...what? Do where? Do what? I am.so utterly confused. Okay so she sees mom and then goes to said academy. She is roomed w 4 other guys that have traits of the 7 dwarves! But no there are only 4. I don't know what they look like and can't get a handle so I made myself a list of their characteristics just so I can remember them. When I have to do that you know it's not going to be fun to read! So, we have a handsome one, silly one, emotional one and standoffish one. Great..original! Shegoes through s couple of events at school that are unprecedented for first year students and has run in w her brother who despises her and makes her go to some wastelands where she could have died! Okay, cool bro! She has some smexy time w only 3 of the me while the standoffs one says I'm out okayyyyy. Oh, I forgot she does make 1 girlfriend and 1 enemy girlfriend bc said girl was w Eldon the cocky handsome one. Deep breath I know this is alot. She learns everyone has magic, weird saying that this professor says creeps her out, some people have 2 powers then boom there's a gauntlet w a screaming banshee mermaid thing! Days go by and raven goes on the last day. Low and behold the stand of fish dwarf helps her out. At no point in time are the like hey raven, you have no magic so how are you gonna survive against a banshee?!?!!? I really hate when characters don't talk about the glaring elephant in the room! Okay so she survives and then there is a formal even to celebrate them surviving! Great family reunion bc they have to sit at the table w the family, this is uneventful bc after diner they run away like children! Then their is another smexy time and I notice the author describes the friday nite and then we are transported to Monday morning every week. Okay cool but when raven said that first weekend I'll do whatever y'all want for Saturday the author missed a great opportunity for raven to I don't know maybe get to know the 4 dwarves! Ask them questions about magic, why are we here, how was school for y'all before coming here, what does it feel like to have magic bc guess what guys I don't have any magic?!?!?!? I'm so tires! She gets a suprise visit from her parents and they told her a seer told them of their 2 kids powers but a suppression was put onto her without theirs and her permission! Uh, why didn't you just tell her that before she went to school so she wouldn't feel unworthy and feel crappy for being a VOID?!?! She's mad and avoids the dwarves for a bit then has a finding a coming excursion at school w the a hole guy and then they finally have smexy time! The author never explains what this coin is for!!!!! Okay, author invents things and then doesn't explain okay cool! Did I mention I'm tired? In bn all this there were a couple of parties, alot of students die from all the years, smexy times and rudeness from a hole brother. Okay, they get swords, raven gets a familiar Griffin! There's a school field trip and the 4 boys and raven are trapped and green masked men attack them and brax, stand off boy is harmed or dead I'm unsure. Raven scream for her griffin and he comes then in ends! Cliffhanger!!!! Beware! Was I going to read book 2 early on? No! Am I going to read it now? Yes bc I have to know what's going on! These young boys are just that young boys and raven and Leila are cool but I'm really interested in what powers raven has and about her griffin. Would I recommend? Nooooo!
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Reviewed in the United States on November 2, 2025
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