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Description
Human MITF 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 capture antibody against Microphthalmia Associated Transcription Factor (MITF). 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 Microphthalmia Associated Transcription Factor (MITF) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Microphthalmia Associated Transcription Factor ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Microphthalmia-associated transcription factor (MITF), also known as class E basic helix-loop-helix protein 32 or bHLHe32, is a protein encoded by the MITF gene. It is a basic helix-loop-helix leucine zipper transcription factor involved in regulating lineage-specific pathways in multiple cell types, including melanocytes, osteoclasts, and mast cells. The term "lineage-specific," as it relates to MITF, implies a gene or trait found only in a specific cell type. Therefore, it may be involved in rewiring signaling cascades that are specifically required for the survival and physiological function of its normal cellular precursors. Along with transcription factor EB (TFEB), TFE3, and TFEC, it belongs to a subfamily of related bHLHZip proteins known as the MiT-TFE family of transcription factors. These factors are capable of forming stable DNA-binding homo- and heterodimers. | |||||||||||||||||||||||||||||||||
| 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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4.5 ★★★★★
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Product Reviews
★★★★★ 5
Comfy back support for lounging
This lounger cushion is very comfortable and supportive and works great in the bed, on the couch, or even the floor when additional back support is needed for reading and other activities requiring you to be propped up.
The size is more suitable to small to average size persons. Larger frame people may find it too small for their needs due to the width and height proportions.
The cushion ships. Compressed into a small package and needs to be fluffed up a bit before use. The one I received expanded fairly quickly and was usable right away, but the instructions say to give it 24 hours to fully expand.
The built in handle makes it easy to drag around to different spots. It also has zippers that allow you to gain access to the inside in case you want to remove some of the fill to adjust the softness to your liking. I found the amount of fill to be perfect as is.
The is an excellent lounging pillow that feels well made, is comfortable, and attractive.
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Reviewed in the United States on November 20, 2024
★★★★★ 5
Solid addition to my bedroom for reading and watching TV.
I ordered this to add some additional comfort in my bedroom for watching TV and Reading. It was amazing how it arrived in a small box and within 2 days, it fully expanded to it's actual size. The cover does have a slight tolerable odor at first. It's not foul or anything, just something I noticed. The smell did go away mostly at the 3 day mark. If you don't like it, I would recommend putting it through the wash once before stuffing it with the Foam fill. The handle at the top has already come in handy with easily being able to move this around. This was a very nice feature of the pillow.
It's grey color blends well with my White comforter and sky blue sheets. This is now providing me with much needed back support and relief. I'm going to order 2 more of these, another one for the same bed and the other for my guest bedroom.
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Reviewed in the United States on November 27, 2024
★★★★★ 5
Very cozy!
I often sleep in on weekends and when I wake up, I stay in bed and watch some TV. I sit up with a pillow propped up behind my back so when I saw this I figured I'd give it a try. I remember having similar ones when we were kids way back in the '60s '70s but this new pillow contains small chunks of memory foam so it is very comfy. The amount of memory foam chunks can be managed by unzipping the well made bottom zipper and removing or adding more memory foam. Although it does not come with extra foam, it is really stuffed full after expanding. It arrives vacuum packed and when I took it out of the plastic it looked like a baby elephant! It does say to let it rest for 3 days occasionally fluffing it to get it to it's total fullness and it did take a couple of days to expand completely but it was well worth the wait!
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Reviewed in the United States on November 18, 2024
★★★★★ 5
You cannot go wrong with this purchase.
Size: Queen (1 Count), Color: Crescent White
I bought this for my husband who was complaining about shoulder pain from rolling side to side nightly. He said the support is great. Quality made and comfortable, a great value for the money. Sleeps better and the company sends extra filling to adjust the thickness if you choose to.
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Reviewed in the United States on April 28, 2026
★★★★★ 5
Thank you Lord!
Size: Queen (1 Count), Color: Crescent White, Size: Queen (1 Count), Color: Crescent White
I appreciate this pillow! Literally helped my neck problems. With the pillows I used before I would have neck pain and would be super uncomfortable. Even to the point I would feel better just sleeping without a pillow. No matter what pillow I used in the house my neck would be sore and would develop headaches.
Ever since I bought this and started using it I haven't had any neck problems. Like none. I love how only one side is cooling so I can decided. The material is really nice and soft. It's also a good hugging pillow. It's definitely weirdly shaped but it has a reason. This thing will probably last me a long time. And I'm glad I spent my money on this!
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Reviewed in the United States on April 11, 2026