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Description
Mouse ALK 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 serine/threonine-protein kinase receptor R3 (ALK) 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 serine/threonine-protein kinase receptor R3 (ALK) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Serine/threonine-protein kinase receptor R3 | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Activin receptor-like kinase (ALK), also known as serine/threonine protein kinase receptor R3 or ACVRL1, is an enzyme encoded by the ACVRL1 gene. It is a receptor in the TGF-beta signaling pathway. This gene encodes a type I cell surface receptor of the TGF-beta ligand superfamily. It shares a high degree of similarity with other type I receptors in its serine-threonine kinase subdomain, a glycine- and serine-rich region preceding the kinase domain (called the GS domain), and a short C-terminal tail. The encoded protein shares a similar domain structure with other closely related ALK or activin receptor-like kinase proteins, which form a subfamily of receptor serine/threonine kinases. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia (HHT) type 2, also known as Rendu-Osler-Weber syndrome 2. | |||||||||||||||||||||||||||||||||
| 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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4.7 ★★★★★
Based on 339 reviews
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Product Reviews
★★★★★ 5
Great product if you know what you’re getting.
Size: 4-tier, Style: wall mount&floor mount, Size: 4-tier, Style: wall mount&floor mount
It’s a great product if you know some details before you purchase. Specifically, for how much it cost it’s a great product. After assembly it is sturdy and will hold anything you’ll be displaying on it within reason. Speaking of assembly, it’s straight forward and intuitive if you’ve assembled other things such as a basic end table.
What you should know: it does not come with instructions. It’s not hard to figure out but if you’re someone that needs instructions this product isn’t for you. That being said, if you see what you bought it’s not hard to look at the picture and assemble it.
It does not come with the shelves. If you can’t buy them premise or make them then this product is not for you.
Lastly, and this part is important to me, not all the pieces are straight after assembly. I used a level to mount the product on the wall and make each shelve straight. However, due to the factory produced screw grooves it is not perfectly straight. If you need it perfectly level looking this product is not for you. Refer to my pictures and you’ll see how the left bar looks crooked even though I mounted it level in every way. However, it gives it a more rustic, modern farmhouse feel that we wanted.
Otherwise, a great product. I am still very happy for what we paid for. I would recommend the product and buy it again.
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Reviewed in the United States on March 6, 2022
★★★★★ 5
Great product!
Size: 4-tier, Style: wall mount&floor mount, Size: 4-tier, Style: wall mount&floor mount
This is my second time purchasing these shelves. They are easy to set up, come with all the screws, brackets, and drywall anchors (if needed). I definitely love the quality, look and versatility. Being that my ceiling is slanted I couldn’t mount them from there but easily mount them to the surface beneath the shelving. My husband loved my idea and how they turned out! Highly recommend!
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Reviewed in the United States on August 8, 2024
★★★★★ 4
No Instructions - Easy to build and study
Size: 4-tier, Style: wall mount&floor mount
I ordered 3, and my wife loves the look of them. They seem to support a decent amount of weight. I supervised my 7 year old (55 lbs) as she scaled a single shelf bracket up the wall after it was mounted, without the planks.
You do need to have some amount of handiness to be looking at buying as it does not come assembled, and does not have the wood planks with it.
A little learning curve I had. Keep the rods only hand snug, not tight, snug, and then as you put it up to the wall adjust as needed to get the heights and widths right to the opposing shelf.
There is also only 8 pipe brackets to hold the shelves down (2 per shelf) and I did not realize this at first and assumed I had 16 (4 per shelf). I had to go back and fix.
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Reviewed in the United States on January 5, 2021
★★★★★ 5
Game changing storage!
Size: 4-tier, Style: wall mount&floor mount
I love the shelves. I got to use my own wood which was a reclaimed piece that I found and sanded and then stained. I'm so proud to see them on the shelves. It got so much clutter corralled from around the room and put in one place where it all looks good displayed together. The support pieces are very good quality and I found this to be very good value for the price. I'm very happy with this item.
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Reviewed in the United States on February 25, 2024
★★★★★ 5
Good all around, some imperfections on threads difficult to screw together
Size: 4-tier, Style: wall mount&floor mount
I received a complete product as advertised, it's a solid steel design with everything you need to mounted included. One small issue I encountered is that the pieces screw together but they wouldn't screw all the way it felt difficult and possibly if forced the steel might crack. Unfairly enough other pieces did completely screw in. So it's probably because of imperfections on the threads.
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Reviewed in the United States on May 9, 2021