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Description
Human Cyclin D1 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 freeze-thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes 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, but avoid repeated freeze-thaw cycles. Pre-Assay Preparation: 1. Remove the reagent 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 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 20ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used 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 cyclin D1 capture antibody. 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 cyclin D1 in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Cyclin D1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Cyclin D1 is a protein encoded by the CCND1 gene. The CCND1 gene encodes the cyclin D1 protein. This gene is located on the long arm of chromosome 11 (band 11q13). It is 13,388 base pairs long and translates to 295 amino acids. Cyclin D1 is expressed in all adult tissues, except cells derived from bone marrow stem cells (both lymphoid and myeloid lineages). Its overexpression has been shown to be associated with early cancer onset and tumor progression, and it can contribute to tumorigenesis by increasing anchorage-dependent growth and angiogenesis through VEGF production. | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenate, cell culture supernatant |
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4.7 ★★★★★
Based on 2272 reviews
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Product Reviews
★★★★★ 5
this book set my soul ablaze! <3
Format: Paperback, Format: Paperback
"i had never really cared about the weather before, but now, clear skies meant everything to me, and i was grateful to see another calm morning."
this book. this book!
i loved the last storm so much. the writing style. the descriptions. the world-building. the characters. the plot twists. the tropes. the sexual tension. the—everything. everything was magic.
the last storm follows our two main characters, ara and rogue, giving us dual POV from both characters (which i loved, btw). ara, a human girl who has been locked away in her father’s estate most of her life, just wants to see the world. all she dreams of is seeing what else is out there. but when her father announces her engagement, she knows that dream will become nothing more than just that—a dream.
rogue, the fae king, is tired of the attacks being rained down on his people. in hopes of finding out the human king adon’s secrets, rogue infiltrates auryna’s borders. in his last resort to gain information, he visits the local pub. to his surprise, the general’s precious only child is sitting at the bar, drink round after round of mead. now he just needs to figure out how to take her without anyone noticing.
first and foremost, let’s talk about the endless list of my favorite tropes and aspects that this book had.
›› enemies to lovers
›› fated mates
›› one bed
›› the chosen one
›› elemental magic
›› actually good and shocking plot twists!!!
›› badass female lead
›› morally-grey love interest
›› fae/human war
›› force proximity
›› touch her and die
›› who did this to you?
›› captor/captive
›› praise k!nk (panting profusely)
“you are entirely the opposite of everything that i am, and i would gladly wear your shackles if it meant i could have you.”
it’s been a long while since i read a book i liked this much. but i just loved this book. it set my soul ablaze. thank you to the author for writing this beautiful story and for blessing me with an eARC! i loved it so much that i immediately bought the paperback upon release!
every aspect of this book was just beautiful. i was blown away by the way the world was described, the way feelings were portrayed, the way the elements were used in the fae’s magic. it just—AHHH! i just absolutely adored it all. i cannot wait for the second book to release next year!
also the way he calls her “little storm” sets my heart on fire.
this was a fast-paced read and if you are a lover of acotar, fbaa, deal with the elf king, or any other similar books, then please stop everything you’re doing and read this book right now. you won’t regret it.
thank you again, jd linton, for giving me the privilege of reading your arc and for blessing this world with the world you created. <3
"something about him pulled me in, like a moth to a flame, and it felt as if i was just waiting for the inevitable burn that came with flying too close to the fire."
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Reviewed in the United States on November 15, 2022
★★★★★ 4
Fun, both heartwarming and heartbreaking
Format: Kindle
Only my second first person written selection, I am still getting used to that aspect, but unlike my first, I enjoyed that the story was told through both MCs. A great enemies to lovers, forced proximity, fated love etc, that resonated to me. There were some small twists that I could see coming, but also a few that I didn’t quite see until the characters were also seeing. Personally, I am more interested in the story than the spice, but with that said, it was well seasoned! I am kind of new to the spice world so I can’t say for sure how this would rate, but it definitely had some heat. I am very glad I happened across this author, and I do plan on also reading the next book….if nothing else, just to see for myself the “transformation” of the characters I’ve grown to love!
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Reviewed in the United States on May 23, 2024
★★★★★ 5
😍😍 BEST DEBUT NOVEL EVER
Format: Paperback
I’m having trouble finding accurate words to describe the way this book made me feel, but I am going to do my best.
To start off with basic elements, the character and world building are phenomenal. I feel a strong bond to not only the two main characters, Ara and Rogue, but to each and every character introduced throughout the book. The author did a stellar job of giving each of them unique personhood. All of the scenes are beautifully described. So much so that throughout the entirety of the book, I could see every scene: the towns, the castles, the meadows, the landscape. I have had difficulty with this and with distinguishing between outlying characters while reading in the past, but I did not have to think to remember details of world or character building because they flowed naturally within the story and were described well. I have read book series before that made me want to be a part of that world, but I actually felt like I got to step into Auryna and Ravaryn!
The plot twists!! Although this is not a suspense novel, it still had me on a rollercoaster of emotions and on the edge of my seat from beginning to end. I haven’t cried actual tears over a book since I was in high school (and I’ve read a LOT). This book finally broke the floodgates in the final few chapters. Multiple times. And we love a good cliffhanger. It truly made me FEEL.
THE SPICE is a solid 3.5/5. Some of the scenes had me flushed, some had me taking notes, some just had my jaw slack and my mouth hanging open. Bravo, JD Linton, bravo.
The relationships: friendships, family, romantic, ALL of the relationships in this book have so much meaning. The author does a great job at making you feel the love, the anger, the peace, the frustrations, the safety, the familiarity, etc. between the characters.
Ara and Rogue. I can not say enough and I also do not want to say too much. Just know that I feel like I know them both, to their core. I know what their childhood looks likes, their darkest moments, their biggest fears, their dreams and passions, what they want in life… The POV switches were seamless. I am so happy this author decided to let us see from both sets of eyes.
I can not wait for book two after that cliffhanger. And there is SO much potential for at least one prequel, I can’t wait to see where this author goes! I hope this series continues and flourishes. Fingers crossed!
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Reviewed in the United States on December 5, 2022
★★★★★ 5
Amazing!
Format: Kindle, Format: Kindle
This book was phenomenal, I devoured it within a few days! For this being a debut novel, it is fantastic and I would’ve thought the author was a seasoned author. I have zero complaints about this book.
Let me start by saying that the world building was phenomenal. I could picture everything in my head because of how detailed it was — that’s how good it was written. And I absolutely love the “captive/captor” trope so much, it’s become one of my favorite tropes, so I was pleasantly surprised to see that this book had that.
I loved the banter between Rogue and Ara — they’re both snarky and witty, plus with the romantic tension, it made the dialogue that much better. Speaking of romantic tension, yes there is spice but not so much of it that it overrides the plot, which I loved. For me, this would probably be on the 3/5 level of spice.
This book had a ton of plot twists and I thoroughly enjoyed it.
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Reviewed in the United States on November 13, 2024
★★★★★ 4
High on Tropes and Satisfaction
Format: Kindle
This is a great Romantasy book full of action, adventure, and everything you look for in this genre.
I won’t lie: it does kinda feel like the author found every common trope from every successful book of this kind and threw them all into this novel. But if it ain’t broke, don’t fix it. Especially in romance, there’s a large audience who has specific expectations, and they want them every time. Nothing wrong with that and many times I’m one of them.
I have no idea what defines a spoiler honestly, so
spoiler alert!!!!!!!
Tropes include:
Only one bed at the inn/bar
Dissatisfaction with life before hunk appears
Lost royalty
The chosen one
Montage of dress up time followed by shocked hunk
Forbidden romance between two from rival peoples
Power that cannot be controlled, simply guided/asked
Gathering intel at the inn/bar
FMC who knows how to fight/use weapons well
There’s probably more but no need to list them all.
Good story and I would recommend!
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Reviewed in the United States on June 14, 2024
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