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Description
Human eIF4E 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 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 eukaryotic initiation factor-4A (eIF4E). 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 eukaryotic initiation factor-4A (eIF4E) 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 initiation factor-4A ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Eukaryotic initiation factor 4E (eIF4E) is a protein encoded by the eIF4E gene. It is involved in directing the ribosome to the cap structure of the mRNA. It is a 24 kDa polypeptide that exists both in its free form and as part of the eIF4F preinitiation complex. Almost all cellular mRNAs require eIF4E for protein translation. The eIF4E polypeptide is the rate-limiting component of the eukaryotic translation machinery and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
Based on 15 reviews
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Product Reviews
★★★★★ 5
Good Quality Cable
Size: 25-Foot, Style: 90° Locking End
Good quality and durable product. We replaced the OEM cable to our RV and have been very happy with its performance.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 20, 2025
★★★★★ 5
Great power cord
Size: 25-Foot, Style: 90° Locking End
I'm very happy with the quality of this power cord. The one that came with my RV came straight out of the side and 50 amp cables are heavy. This cord has a 90 degree end that takes away the leverage factor of the other one. This should be much easier on the RV wall connector. Well worth the cost.
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Reviewed in the United States on May 1, 2024
★★★★★ 5
Good RV Power Supply Cord
Size: 25-Foot, Style: 90° Locking End
It's an overall good value for the cord. I purchased so I'd have a spare in the rv and because my original is very short, I may need the extra length one day. I plugged it in and it works as intended, it appears to be good construction and quality materials.
I've been caught in a situation before where my cord went bad (the inlet on the camper overheated and caused the plug in the melt and burn up, this could happen to anyone) it was after hours, so I couldnt run to a store and get one. Had to suffer through the night with no AC and wait until the next morning to go to an RV superstore and pay double what Amazon charges. If you take your rv out alot I highly recommend keeping a spare cord.
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Reviewed in the United States on July 16, 2020
★★★★★ 5
Great product! I do not understand why anyone would make or buy any cord without a 90 degree plug-in for their RV.
Size: 25-Foot, Style: 90° Locking End
We had to replace the plug on our RV: The plug was destroyed by the stiff 50 amp cord that; came with our RV that put a lot of pressure on the plug-in on our RV causing it to arc and melt both the male and female where the cord was plugged into the RV. . The heavy duty 90: degree end has a soft flexible cable and greatly reduces the stress on the RV plug. I expect this cord will last much longer. Would recommend this cord to anyone. I do not understand why anyone would make or buy any cord without a 90 degree plug-in for their RV. Dealers may not have this item in stock.
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Reviewed in the United States on July 14, 2018
★★★★★ 4
Good compromise
Size: 25-Foot, Style: 90° Locking End
I decided to go with this much cheaper version of the same yet higher quality cord from camping world. I really like the 90 degree connectors as these cords are heavy and I did not like the added stress on the RV power connector of the straight connectors. Also did not like the way the straight connector sticks out on camping would’s version so if I accidentally bumped into the cord it could easily be broken. The clamp ring on this one has large lugs making it much easier to tighten onto the RV threads. I really struggled with the ones that don’t have the large lugs. The easy to grip handles are also nice to have when removing from power. My one complaint, the cable jacketing is of lesser quality. You can literally feel the individual conductors inside the jacketing when handling. This makes me feel like this cord will not have the sturdiness to last over time and an insulation failure could be a dangerous situation. If I could get the connectors of this cord with the cable quality of the camping world version, I would pay for the quality.
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Reviewed in the United States on June 9, 2023