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Description
Mouse CTTN 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 cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold PBS and resuspend 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 for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 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 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 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 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 cortactin (CTTN) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of cortactin (CTTN) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Cortactin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Cortactin (derived from "cortical actin-binding protein") is a monomeric protein located in the cytoplasm that is activated by external stimuli to promote polymerization and rearrangement of the actin cytoskeleton, particularly at the cell periphery. It is present in all cell types. Upon activation, it recruits the Arp2/3 complex to existing actin filaments, promoting and stabilizing actin branching nuclei. Cortactin is important for promoting lamellae formation, invasive cell formation, cell migration, and endocytosis. Diseases associated with CTTN include sigmoid sinus adenocarcinoma and Wiskott-Aldrich syndrome. Pathways associated with CTTN include developmental Gap-Rob signaling and Syndecan-3-mediated signaling events. Gene ontology annotations associated with this gene include profilin binding. An important homologue of this gene is HCLS1. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.7 ★★★★★
Based on 2061 reviews
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Product Reviews
★★★★★ 5
A Charming Historical Cozy Mystery
Format: Audiobook
Lady Hardcastle and her companion Florence Armstrong, have moved to the country. They think they’ll find a quiet life, but almost as soon as they set foot into the area, things start happening. Before they know it, they’re involved in helping to investigate two very different murders. They have some previous experience with such things, but will the constabulary take them seriously? I mean, they are women, after all; what cam they possibly know about such things?.
This was a charming cozy mystery, and I can’t wait to listen to more books in this series. The writing was top notch, and the characters a delight. The plot was beautifully developed and executed. All in all, this was a jolly good read. I really enjoyed learning some of the backstory for Lady Hardcastle and Flo.
I listened to the Audible audiobook of A Quiet Life in the Country. I thought it was remarkably well narrated. Truly, I look forward to learning more about Lady Hardcastle, Flo, and their new friends and associates.
I purchased a copy of this audiobook from Audible or Amazon. I can’t remember which. In any case, all opinions in this review reflect my true and honest reactions to reading this book.
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Reviewed in the United States on February 8, 2024
★★★★★ 4
What a delight to read!
Format: Kindle
I'm not quite sure what I expected when I plunked down my money and downloaded A Quiet Life in the Country. A modern variation of the Golden Age country house mystery? If that was it, I didn't get it. What I did wind up getting was a whole lot more. I will warn you ahead of time: if you are a fan of those classic mysteries, T.E. Kinsey's book may not be your cup of tea, and now I'll set about telling you why.
Kinsey has written a good, puzzling, multi-layered mystery that gives you a real feel for village life out in the English countryside at the turn of the last century. While the plot and the setting are first-rate, it's Kinsey's characters that make this book shine-- and what makes the book turn away from those classic mysteries.
Inspector Sunderland moves from being a stereotype to being someone rather special, which I always love to see (being sick to death of incompetent policemen in the books I read).
Lady Emily Hardcastle's background is very mysterious indeed, and I hope it takes several books to have all the details teased out for us to see. However, it is the relationship between Lady Emily and Florence that is the pièce de résistance of A Quiet Life in the Country. Their dialogue is so full of mutual admiration, wit, and humor that it is, quite simply, a delight to read. As Sunderland witnesses one such conversation, he asks, "Are you two a music hall act?" and you have to admit that he has a point. (If they were a music hall act, I'd pay to see them.)
If you're in the mood for something that's light and fun, something that teases your brain, takes you to the English countryside of a century ago, and makes you laugh, have I got a recommendation for you: T.E. Kinsey's A Quiet Life in the Country. I'm on to the second book!
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Reviewed in the United States on March 6, 2017
★★★★★ 3
are looking to settle in to the nice, quiet
Format: Paperback
Welcome to Littleton Cotterell,a small village in Glouchestershire. The year is 1908, and Lady Emily Hardcastle and her intrepid maid, Florence Armstrong, are looking to settle in to the nice, quiet, retired life after several years spent adventuring around the world.
Naturally, such plans are always disrupted. In this case, the two women discover a dead body hanging from a tree while out on a morning walk. The death is meant to appear as a suicide, but certain inconsistencies seen to point more towards murder. With the local constabulary over their heads, Lady Hardcastle and Flo must draw on their previous experiences to solve the murder before anyone else gets hurt.
The mystery goes off in fairly expected fashion, with the eccentric and kindly Lady Hardcastle relying on her witty and resourceful maid. There are suspicious characters and red herrings aplenty. And, naturally, the initial mystery gets wrapped up in several others in the course of the book.
I will say this for A Quiet Life in the Country: it does not take itself too seriously. The usual tropes of the cozy mystery are addressed with a wink and a nudge (such as one character explaining to Lady Hardcastle that the tiny, 30 person village she just moved to is actually the murder capital of the country). I appreciate the effort made to acknowledge all the commonly used bits that go into a cozy murder mystery, and it certainly helped to dispel a feeling of deja vu.
In all, if you’re looking for a nice, quick mystery with not one but two strong female leads, you could do far worse than to join Flo and Lady Hardcastle on their adventures.
A free copy of this book was provided via Goodreads Givaways in exchange for an honest review. A Quiet Life in the Country is currently available for purchase (and, at the time of this writing, free via Kindle Unlimited).
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Reviewed in the United States on November 23, 2016
★★★★★ 5
Pay no attention to Amazon's forced "one word" reviews. Read this review instead!
Format: Kindle
When led to this page for my review I found a series of leading questions which I found quite offensive. Don't put words in my mouth Amazon. I'm quite capable of giving my own ideas and views! For instance instead of the above four one word clap trap, I give you my review: "Full of surprises" indeed!
That being said, I absolutely adored "A Quiet Life In The Country". I take full advantage to read for free lots of book through Prime, but most of it is schlock. This is the gold piece found buried deep, and worth the search. It's fun and frothy reading at the level of P. G. Wodehouse and Georgette Heyer. I echo a hearty Amen! to those who found Flo and Lady Hardcastle the most diverting sleuthing duo to come along in ages. I want much much more! Well researched too, as I learned who in the upper crust sails (as in Ladyship) and an assortment of early 20th century sports figures like W. G. Grace and Jack Hobbs, and after googling pictures of same, got quite a giggle out of Grace being portrayed as Oscar Wilde.
This is actually my second read of this series, and it is even better than I remembered. In the second one you get even more behind the scenes with Lady H and Flo and it is a wild ride. These are first class offerings and I'm actually buying them all and can't wait until the newest comes out next June (2017)!
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Reviewed in the United States on January 3, 2017
★★★★★ 4
Well written. Funny...out loud gafawing funny. Fun. Well crafted plot. Wonderful characters.
Format: Kindle
What a lovely surprise!
A well written, well plotted. Funny. Fun. Mystery with two fresh and fine friends...Lady Hardcastle and Flo Armstrong , her erstwhile Lady's maid, Servant, Strong Right Hand Woman and friend...and...Inspector Sunderland, who finds the two so innovative and essential to the investigation of Two Murders and Some serious thefts that he is determined, and says so, that whenever they can he desires them to assist in future!
I so enjoyed this first book in the series I invested in the next four...so far!...in the series.
What fun...the women and now the Detective Inspector, as well, have such comical and engaging exchanges one finds herself (me) laughing so heartily she needs to pause and wipe her eyes repeatedly as she reads the story!
Would highly recommend this series to anyone who loves a good, well plotted mystery and a giddy, heartfelt laugh...well, series of laughs, at the same time.
Dialogue is often funny...though murder and mayhem are not!...and this is a careful blend of both....just sayin'.
What a wonderful edition to a huge genre of cozy and some not so cozy mystery books.
I recommend this series.
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Reviewed in the United States on June 7, 2024