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Description
Human GFRAL 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: 1. 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: 1. 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. 2. 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. 3. 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. 4. 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. 5. 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. 6. 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 a capture antibody against GDNF family receptor alpha-like (GFRAL). 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 GDNF family receptor alpha-like (GFRAL) in the sample. The absorbance (OD value) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human GDNF family receptor alpha-like ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | GDNF family receptor α-like is a protein encoded by the GFRAL gene. An important analog of this gene is GFRA2. GDF15, a brainstem-restricted receptor, regulates food intake, energy expenditure, and body weight in response to metabolic and toxin-induced stress. After interacting with its ligand, GDF15 interacts with RET and induces cell signaling by activating the MAPK and AKT signaling pathways. | |||||||||||||||||||||||||||||||||
| 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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4.4 ★★★★★
Based on 514 reviews
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Product Reviews
★★★★★ 5
Absolutely top-knotch
Format: Kindle
9.5/10
This is the pinnacle of Star Wars comic books. A great way to tie in their Indiana Jones character in Aphra and the mainline series to tell an amazing story. Only complaint is a couple of the issues artwork I was not a fan of. I like the more realistic look. Just make sure you read Aphra book 1 and the previous SW books to understand it better. Aphra book 1 being more important
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 6, 2018
★★★★★ 5
Star Wars embraces fantasy
Format: Paperback
This is Star Wars at its' strangest, and that's a very good thing. Luke and co. fighting through what could easily be Dracula's castle is a truly unique experience. I don't wish to say more for fear of spoilers.
As a note though you will get more out of this if you've been following the Star Wars and Dr. Aphra comics. However you can get by without that knowledge as well.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on October 26, 2017
★★★★★ 3
Gothic Star Wars
Format: Paperback
This trade paperback collects all the issues for the Screaming Citadel story spread over several titles, including the main stay Star Wars series and the Dr Aphra book. As one might expect from a story spread over different titles with different artists and writers, the presentation varies. The art is all over the place. In the Marco Checchetto-drawn initial issue, everyone’s favorite amoral artifact hunter, Dr Aphra, is a striking space vixen. But in the following issues she’s hardly recognizable as the same character--mousier, if still menacing, in her trademark Russian tanker’s hat. To a lesser degree, the same is true for the other characters, including the main SW group. It’s understandable, but a bit disconcerting.
The story centers on Dr Aphra, who, in need of a Jedi for one of her typically nefarious purposes, recruits Luke into her scheme. Unfortunately for Aphra, she’s up against a more ruthless foe in the harlequin-looking vampire-like Queen of the Screaming Citadel. Before long, the rest of the group has to show up to rescue them. It’s a gothic story, set in scary castle—not the usual Star Wars fare. There are some good points. Dr Aphra’s almost sociopathic outlook is always good for a few choice lines, the “murderous machines” Bee Tee and Triple Zero are on hand for their own gruesome commentary and some of the Queens hench-people, while not given much to do, are interestingly designed. But overall, the horror movies plotline didn’t seem much like Star Wars to me. Recommended for those who enjoy that type of story, or completists.
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Reviewed in the United States on February 27, 2018
★★★★★ 4
Luke and Doctor Aphra team up!
Format: Paperback
This is the second crossover event in the Marvel Star Wars comics. It brings the ongoing Doctor Aphra and Star Wars series together. I liked the pairing of Luke with Aphra. They play well off of each other with Luke's naive goodness and Aphra's experienced gray morality. I liked when she called him a wannabe padawan.
There are some well designed characters in this comic. The residents of the Screaming Citadel have a goth bdsm vibe. Luke even gets to dress up. I liked seeing him in something different.
I want to know more about Sana and Aphra's past!!! Please, Marvel, make a queer love story prequel!!!
The murder droids are wonderful. Having them on the same side as the "good guys" for at least the time being led to some funny situations.
The last panel intrigued me. I give this graphic novel a 4/5. I am always here for more Doctor Aphra!
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Reviewed in the United States on December 29, 2017
★★★★★ 5
Excellent mini series.
Format: Kindle
This is an excellent follow up to Vader Down. Luke Skywalker and friends take on a bigger threat than The Empire and Darth Vader that is connected to the Jedi. Luke and Dr. Aphra join forces to find the answers Like is seeking. Truly worth reading and entertaining.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 23, 2019
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