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Description
Rat 5HT4R 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 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 5-HT4 receptor (5HT4R) capture antibody. 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 5-HT4 receptor (5HT4R) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat 5-HT4 Receptor ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Serotonin receptor 4 (5HT4) is a protein encoded by the HTR4 gene. This gene is a member of the serotonin receptor family and a G protein-coupled receptor that stimulates cAMP production in response to serotonin (5-hydroxytryptamine). The gene product is a glycosylated transmembrane protein that regulates the release of various neurotransmitters in the peripheral and central nervous systems. Multiple transcript variants have been described, encoding proteins with varying C-terminal sequences. | |||||||||||||||||||||||||||||||||
| 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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4.8 ★★★★★
Based on 24 reviews
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Product Reviews
★★★★★ 3
Solid example of 70's filmmaking
I had forgotten that I had this film in a pile to "watch later" and finally did and was glad I did. This overlooked 1972 film from director Michael Richie while short on run time and story development is still a worthy viewing experience.Mr.Richie went onto a fine career doing such films as The Candidate,The Bad News Bears,Semi-Tough and a few notable comedies like Fletch and Wildcats.He also did Student Bodies which he had his named removed from so nobody's perfect.The film has a great cast it stars veteran tough guy actor Lee Marvin as Nick a mob fixer who is assigned a job to reel in a renegade hood. This hood is a guy named Mary Ann( I kid you not) and he runs a drug and white slavery ring out in rural Kansas City.Played to the cusp of over acting by Gene Hackman Mary Ann uses a meat packing/slaughterhouse plant as a front for his illegal activities.Mary Ann has a dim witted brother called Weenie(at least not Ginger to his brother Mary Ann thankfully) who acts as the muscle played by Gregory Walcott.Nick is a city slicker based out of Chicago who is quiet,mean looking and well dressed.Mary Ann is loud and obnoxious and a country boy .Mary Ann has angered the Chicago mob by stopping his payments and going rogue.He's also grounded up previous gangsters from Chi-Town who were sent to persuade him into sausages and sent them back to Chicago in bundles of meat.So Nick ,a trusted sidekick driver and 3 "green" young wannabees head from Chicago to Kansas City to see what can be done to correct the situation.What you get besides a lot of violence is the classic city versus country scenario. Slick Nick and company pop into Mary Ann's barn where various young girls are being drugged and penned like pigs or cows would be.One of the girls is played by Sissy Spacek(you might have heard of her) and another is played by 70's B movie starlet Janit Baldwin( Gator Bait,Ruby) .Nick is not amused by MA's little gambit and informs him that he's taking one of the girls as down payment for what he owes.He chooses Poppy (Spacek) a pretty little blond who along with Violet was raised in a Missouri orphanage .Said orphanage also was a front for breeding future girls to be sold as sex slaves.MA says that he'll make good on the money he owes but of course you KNOW he's not going to.Violence erupts Nick and Poppy are on the lamb and the rest of Nick's associate's are either killed or on the run as well.Nick and Poppy have a memorable scene in a wheat field where a hired hand tries to grind the two up with a giant wheat harvester .This scene is the most memorable thing in Prime Cut.You feel the fear of 2 people lost in a wheat field and a giant combine machine trying to rip them to shreds.The film has great cinematography and the colors are top notch.Something about 70's cinema and the whole style of shooting makes it stand out.As a last twist in the story Mary Ann is married to Clarabelle (Angel Tompkins) who once was involved with Nick.Will she aid Nick in bringing down her husband or stay loyal to him? This film could've used more story building and feels rushed at times.It's also the film debut of the young Sissy Spacek who's good here.She play's naive and resourceful very good here.Poppy her character reminds me of the character played by Jennifer Jason Leigh in Miami Blues. Both have that "are you kidding me" sense of naiveness that makes them endearing.Baldwin also made her debut here and is less memorable but fine for what's asked of Violet.To sum it up if you want a fine nights viewing I recommend Prime Cut.The cast is stellar ,the look awesome and the action intense.
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Reviewed in the United States on December 25, 2011
★★★★★ 5
An important restoration
These three films contain so much beauty, and so much heartache, it's hard to know where to begin. First off, if your a ciniphile, or criterion collector, it's pretty much a must to own these newly restored films. I believe the original prints where very damaged , or completely lost in a fire, so the restoration is nothing short of amazing. The films do look older then they are, but the clarity and visual beauty is not diminished here at all.
The films basically follow a young boy Apu throughput his life, growing up Bengali in India , and the trials and tribulations he faces from his poor rural behinings, to his education and later move to Calcutta.
On my initial viewing of these films, I thought the heartache was too much, and quite honestly put them out of my mind, but they actually never left, due to the power of them, and the vision of this fantastic director, and the talented actors. Upon reviewing them, at least 3 times each, I realize there is much beauty and hope, along with the pain..in these quite frankly incredible films.
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Reviewed in the United States on August 21, 2021
★★★★★ 5
One of The Undeniable Masterworks of Cinema
This extraordinary trilogy earns it's place among cinema's greatest masterworks. The saga a young Bengali boy's journey into adulthood, feeling his way through the mysterious world, as he experiences living in rural poverty, trains to be a priest like his father, attends university in Calcutta, the unusual circumstances that he meets the love of his life, and becoming a wandering writer. Filmed in an unpretentious neorealist style, simply told but complex in nature, life, loss, love, family, religion, and one's place in the world are all confronted with uniquely gentle and artistic charm and heart breaking tragedy. These films are so pure, so genuine, and so thoughtful, they reach the heights of humanity in cinema. This towering landmark in Indian cinema was a shot heard around the world when first released in the 1950's , winning awards, establishing Satyajit Ray as a world class director, popularizing the music of Ravi Shankar, and has enchanted film-lovers ever since. This Criterion Collection set is superb. Extra content includes plenty of interviews, documentaries, and essays. The restoration is excellent -nothing short of a miracle, considering the original prints were damaged in a fire (this is documented in one of the many extras). These are must-see entries in world cinema and highly recommended to fans of foreign film (especially Kurosawa and Italian neorealists) and those that appreciate great storytelling.
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Reviewed in the United States on August 11, 2018
★★★★★ 5
Terrible delivery
This review is about product delivery.
The Apu Trilogy is one of the best trilogies in the style of Italian neorealism ever made. These movies are international treasures and should be preserved for posterity. I wish Amazon packaging had considered that.
When Amazon sends me a single battery, it comes wrapped and boxed in a giant box. Almost everything comes in oversized packages, but they sent the three DVDs without any protective envelope, box, or sleeve. It arrived in its original packaging, dinged on all four corners. The DVDs were a birthday gift. Extremely disappointed!
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Reviewed in the United States on August 11, 2023
★★★★★ 5
Stunning Document of Humanity
I waited far, far too long to immerse myself in the films of Satyajit Ray. After finally watching the Apu trilogy on The Criterion Channel, I immediately purchase the Criterion set (because the internet's going down someday and these are pantheon-level, must-have films).
Ray's 3-film trilogy following the arc of a boy's life from his idyllic rural village in India to the teeming metropolis of Calcutta covers just about every aspect of human growth and conflict. Family, curiosity, mischief, tragedy, love, desire, intellectual pursuit, and ultimately, the responsibility of parenthood all unfold in Ray's epically framed cinema. At times one feels as if watching a documentary, glimpsing the intimate moments of lives we might never otherwise see. Ray's direction lets every scene breathe fully and the actors feel perfectly natural. It's also a real treat to hear the early works of a young Ravi Shankar in his perfectly timed soundtrack utilizing both traditional and modern treatments of Indian music. Ray's Apu trilogy--as well as the rest of his film catalogue--are must-watch movies for any student of film or anyone who appreciates a beautiful, heartfelt, and philosophical portrait of people enslaved by pleasures, responsibilities, and tragedies.
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Reviewed in the United States on August 24, 2021
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