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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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4.4 ★★★★★
Based on 1654 reviews
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Product Reviews
★★★★★ 4
Good Short Read
Format: Kindle
I found the story to be pretty engaging, but I felt like there could have been more depth to the story.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 25, 2018
★★★★★ 4
Beautiful Family Contemporary
Format: Kindle
“But I believe strongly that we all have multiple versions of ourselves. And the true test of love is learning to accept all of those versions, even when it’s messy.
Actually, especially when it’s messy.”
I LOVE family-centric contemporary stories. They are so easy for me to get invested in and I love seeing the relationships form and evolve. I just love them so much. Here We Are Now was a really good family centric contemporary, that also really highlighted opening yourself up and conquering your fears - whatever they may be. We follow Taliah as she meets her rockstar dad Julian Oliver for the first time, when he asks her to come visit his dying father. Tal learns more about her mom, Julian, and herself than she ever expected and she has to learn to reconcile these new truths with what she’s always believed to be true.
Things I Liked
I really loved the flashbacks we get throughout the story. They show personality, relationships, and I love that we get to see more of Lena’s Jordanian culture. They really helped develop the characters and provide more backstory and depth.
I really liked a lot of the characters! I thought Harlow was a great friend, who tried to help Taliah grow and open up about things, even if she was uncomfortable. Debra, Tal’s grandmother is so kind and insightful and warm.
I also liked the moments we get to see Tal and Julian learning more about each other. These pure family moments are the ones that really shined for me and gave life to the story. I also really liked that they bonded over music!
Things I Didn’t Like
I have kinda mixed feelings about Taliah. I understand that Tal’s been put in this weird position and has a lot of confusing and probably conflicting feelings, but I thought she was being purposefully difficult a few times. But she did apologize for that and for being hard to get to know - and I liked that. I think overall I was just a little indifferent to her, which was unfortunate.
I feel like the budding romance between Tal and Julian’s neighbor Toby, was pretty unnecessary, and mostly just took page time away that could have been used to further develop family moments.
This such an easy book to get invested in. I loved seeing the family moments and Julian and Tal becoming closer, and while I would have liked more development in the family relationships, I was satisfied with what I got. Here We Are Now is a lovely story of family and discovering where you fit in.
I received a copy of the book from Balzer + Bray via Edelweiss in exchange for an honest review. All quotes are taken from an ARC and are subject to change.
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Reviewed in the United States on November 7, 2017
★★★★★ 5
Ahhhh!
Format: Kindle
So I went back and read the first Dreamers Bay book again before reading this one. So very good! Melissa never disappoints! Kyle and Savanna's story is so great. It is funny, spicy, and oh so sweet and getting to revisit Devyn and Elizabeth again just makes me smile! On how I wish Ms. Brayden would revisit the girls from Soho series. Hint, hint! They were the first books I ever read from this author and they hold a special place in my heart! I have probably reread then 3 or 4 times each. It would be so great to see what Hunter, Sam, Brooklyn, Jessica, Hope and Mallory are all up to in the future. As readers we get invested in this wonderful authors characters and getting to catch up with them is like getting an early Christmas present! Buy this one - you won't regret it and if you have never read Melissa Brayden's work before you are totally missing out!!
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Reviewed in the United States on July 28, 2025
★★★★★ 4
Small town romance. Love the banter.
Format: Paperback
Savanna Potter is a perky can do person that is beloved in her small hometown. But she has suffered a lot of losses including her parents and the aunt who took her in and raised her. On a weekend in Charleston she has a chance meeting and hook-up with the right person but at the wrong time. They agree to meet up again in a year but Dr. Kyle Remington doesn’t show up. A few months later Kyle arrives in Dreamer’s Bay. She is embracing small town living and wanting Savanna as well.
Brayden is delightful, as usual, with her witty banter. This is a fairly light and breezy LesFic romance. There are some side plots. But the easy romance between Savanna and Kyle is the focus. I think the only extra thing I wanted was to know more about Kyle. Everything is from Savanna’s POV and you see Kyle mostly in Savanna’s world. It was almost as if her character was irrelevant to the plot other than for her work crisis. Why did she change careers? Does she have a family that she stays in touch with? But with that said this is the kind of book I pull easily from my shelf to read again on a day when I want a quick pick me up.
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Reviewed in the United States on May 20, 2025
★★★★★ 5
You'll laugh, you'll cry, you'll want donuts...classic Brayden
I enjoyed several things about Dream a Little Dream. The small-town setting, checking in with folks from a previous book - Beautiful Dreamer - in this case -
how the author focused on the relationships Savanna and Kyle have with their friends/families, which helped me have a better understanding of each character. In my opinion, Brayden is one the best out there writing dialogue between all characters in her books. The small details she adds to each person help you get to know them a bit better. And the story took a few "did not see that comings," which kept me turning the pages long after I should have turned off the lights.
But what I enjoyed most was simply Savanna and Kyle. They have rocketed to second place on my "Top Ten Melissa Brayden Character" list. Both go through several personal and professional ups and downs, but it is Savanna who makes me want to reach into the book and give her a big ole "everything's going to be okay, hug."
It's classic Brayden, with a twist.
I give it a solid 12.3 out of 10. I highly recommend it.
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Reviewed in the United States on May 23, 2025