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Description
Human EIF2aK3 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. In order to further lyse tissue cells, the homogenate can be ultrasonically disrupted or repeatedly frozen and thawed. Finally, the homogenate is centrifuged at 5000×g for 5-10 minutes, and the supernatant is taken for detection. Cell lysis solution: Adherent cells are gently washed with pre-cooled PBS, then digested with trypsin, and the cells are collected after centrifugation at 1000×g for 5 minutes; suspended cells can be directly collected by centrifugation. The collected cells are washed 3 times with pre-cooled PBS, and 150-200uL PBS is added to each 1×10^6 cell for re-suspending (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and the cells are broken by repeated freezing and thawing or ultrasonication. The extract is centrifuged at 2-8°C and 1500×g for 10 minutes, and the supernatant is taken for detection. Cell culture supernatant: Please centrifuge at 1000×g for 20 minutes, take the supernatant for detection, or store the supernatant at -20°C or -80°C, but repeated freezing and thawing should be avoided. 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 capture antibody against Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (EIF2aK3). 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 level of Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (EIF2aK3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2aK3), also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), is an enzyme encoded by the EIF2aK3 gene. The protein encoded by this gene phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (EIF2), leading to its inactivation, which in turn rapidly reduces translation initiation and inhibits overall protein synthesis. It is a type I membrane protein located in the endoplasmic reticulum (ER) and is induced by ER stress caused by misfolded proteins. Patients with mutations in this gene develop Wolcott-Rallison syndrome. | |||||||||||||||||||||||||||||||||
| 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, cell culture supernatants, and other biological fluids |
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4.1 ★★★★★
Based on 5 reviews
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Product Reviews
★★★★★ 5
A beautifully drawn fun book.
Format: Paperback
This was an extremely fun book that works as another chapter in the Energon Universe Image is building. The art work is fantastic and the story is full of action and is told in a fast pace. Another reviewer complained it is woke and forced diversity but I did not see that at all. If you love GI Joe you will love this book.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 8, 2024
★★★★★ 4
A great pick up for new Joe fans
Format: Paperback
A very fun read! I’m a fan of Kelly Thompson in general and this did not disappoint. I recommend it if you liked Thompsons other work. It’s a bit reminiscent of her Black Widow run.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 19, 2025
★★★★★ 1
Shoehorned Diversity
Format: Paperback, Format: Paperback
Two beloved female characters who are decades old turned gay. Not the gift I was looking to give a new generation of Joe fans.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 6, 2024
★★★★★ 1
Please keep Kelly Thompson away from G.I. Joe!
Format: Paperback
I only read this because it was one of the mini series that was setting up the new Energon Universe of GI Joe. The other minis were quite good and I hoped this one would be the same. Nope. It’s terrible. Terrible dialogue, terrible characterization and terrible reimagining of several popular characters. The plot is bare bones, poorly executed, and often so contrived and cliched I'm convinced Thompson must have watched a bunch of ninja movies from the 80s before writing this and thought she was being clever for ripping them off. The art is passable but the artist definitely needs to work on his fight choreography and stop drawing women so jacked. Scarlett and Jinx look like they have been taking steroids. And let’s not forget that Thompson loves writing lesbians, so it’s heavily implied Scarlett and Jinx are a couple. Because that’s what modern audiences want to see, more historically hetero characters made gay. Thompson, like Tom King, needs to be kept far away from popular characters and just stick to creating her own original stories where she can work out her issues and fetishes away from the rest of the mainstream comic reading public. And if I ever see her name on another G.I. Joe comic I will most definitely avoid it like the plague. Just my two cents.
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Reviewed in the United States on February 22, 2025
★★★★★ 1
Painful Read
Format: Paperback
This is a painful read. I only got it because I’m reading the rest of the EU Booms.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 16, 2025
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