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Description
Mouse MANF ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold PBS and resuspend 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 1mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 1000pg/mL). Then dilute to the following concentrations: 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.25pg/mL, 15.625pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 1000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 500pg/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 mesencephalic astrocyte-derived neurotrophic factor (MANF) 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 mesencephalic astrocyte-derived neurotrophic factor (MANF) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Mesencephalic Astrocyte Derived Neurotrophic Factor ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Midbrain astrocyte-derived neurotrophic factor (MANF) is a protein encoded by the MANF housekeeping gene. It is a highly conserved protein with known functions. The protein was initially thought to be elongated at the N-terminus and contain an arginine-rich region, but transcriptome evidence suggests a smaller open reading frame that does not encode the arginine tract. A specific mutation, altering codon 50 from ATG to AGG or deleting it, has been reported in various solid tumors. With the revision of protein size, this codon is now identified as the start codon. | |||||||||||||||||||||||||||||||||
| 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 | 15.6-1000 pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.2 ★★★★★
Based on 9 reviews
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Product Reviews
★★★★★ 5
Lightweight, Options, and Ease of Use Make this a Great Watch
Color: Black
I have purchased many Timex watches over the years and I am entirely satisfied with this watch. I always buy the sports watch, as I swim and do triathlons.
Things I like about the watch:
*Setting the watch is easy and the instructions are clear (although the printed instructions are very small!)
* The watch is not too big. So many watches have too many features and they are large, bulky, and heavy. This watch is sleek, has a low profile, and has a size that is ideal for average needs.
* The numbers are big enough to see easily at a glance.
* There are enough useful options, and this watch is not overloaded with too many options. Too many options are confusing to me. I like the simplicity of this watch and the ease of use of the options it has.
* I like the plastic watch band. I have tried the velcro strap bands and they wear out after a year or so of use. The plastic band will eventually break, but is fairly easy to replace.
* I like the price. At less than 35 dollars I have everything I need in a watch.
* I like and use frequently the Indiglo feature. I have even used Indiglo to find my way to the bathroom at night from bed! The light on the dial is bright enough to make your way through the dark for a short trip!
Things I don't like:
There is not much I don't like about this Timex watch. I suppose my biggest complaint about this watch and any Timex watch is the difficulty in changing the battery. The battery will eventually need replacement, and of course one has to take off the back of the watch to replace the battery. This is tricky and takes a steady hand. I wish Timex would make the battery more accessible so changing the battery would be as easy as sliding one in or out of a hinged door (like on a hearing aid).
Obviously, there is much more to like about this watch than not like. I do recommend it for a good sport watch. It will withstand chlorine pools, lake water, rainfall, high and low temperatures, but be careful not to scratch the dial window! I scratched the main window crystal and ruined my last watch. Avoid spraying any kind of bug repellant on the window as well, as this can mar (dull) the surface.
For me, this watch meets all my needs and I give it a 5-star rating. My needs are faily simple. There are certainly other watches that offer more features, but to me, the added features add complexity and are not worth the added bother.
The price is good. The watch is good quality. This is a good buy.
Jim "Konedog" Koenig
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Reviewed in the United States on May 23, 2008
★★★★★ 4
Great watch; but why the triangle screws?
Color: Black/Digital Gray/Silver-Tone
I have been using these watches for many years with very good luck, and would normally give them 5 stars. They are comfortable, waterproof, durable, and have an alarm and a countdown timer. (The waterproof and countdown timer - that can be set to repeat - make them good for sailboat racing, and these are much cheaper than sailing watches. )
But at some point before the watch breaks, the battery will die. It is not hard to replace the battery. Except now the watch comes with triangle head screws holding the back on, rather than the regular phillips head screws they used to have. Why? Is it that important to keep people from opening up their watch? Making things harder to fix is a step in the wrong direction. Knocking off one star for that.
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Reviewed in the United States on August 28, 2025
★★★★★ 5
Still the champ.
Color: Black/Negative Digital/Gray, Color: Black/Negative Digital/Gray
I absolutely LOVE my Casio GBA800 that I have bully bars on, so she's quite huge. Analog with Bluetooth, pretty cool. If I ever remember to use such things. I have a phone and laptop on me usually, why do I need to play with my watch? I want to tell time, that's it.
Comes along a good sale for an Ironman, and I used to be a complete fanatic for only Ironman for a real person's needs. Why not, I've been fighting the Casio all day with my coat cuff, let's get something a bit slimmer for when it's coat season. See if they're still great. Yup! Sure is.
It's been probably 25 years ago last time I wore this style of Timex, and can you believe the button setup and beeps are all still the same as way back when! And somehow, my subconscious remembers the button presses. Wild.
This thing is light as a feather, and I got the inverse LCD for fun, which is cool as all get out, but takes some getting used to. The Indiglo sure looks awesome, though, and she's like Knight Rider down there. Stealth. The display is big, and easy even for my old eyes, the band is supremely comfortable, and there's a strap catch that actually works correctly! With no Bluetooth, battery life should be outstanding - always used to be. Back when watch batteries were like $12 each. There goes my allowance! Lol.
I'm very impressed by this timepiece. I really didn't mean to order order it, but, oh well. I love it as much as my Casio. Affordable tough watches rock!
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Reviewed in the United States on January 21, 2025
★★★★★ 5
The colour is cool
Color: Black/Digital Black/Black
Cool stuff
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Reviewed in the United States on May 20, 2026
★★★★★ 5
This Timex Ironman lives up to its name
Color: Black/Digital Gray/Black
Timex Ironman watches have been with me for a decade. This particular one I just ordered as a backup for the same one that I have had for seven years (and still works perfectly). I don't know why I ordered a backup except that I love it so much that I couldn't fathom the idea that Timex might discontinue this model. Why do I love it so much? Because it is functional in many ways. Aside from its durability, reliability and the fact that it fits on my wrist so comfortably, it is easy to read and extremely useful for tasks that need countdown alarms as well as regular timers (it has three which you can set). Since I gave up "fashionable" watches years ago in favor of practical, easy to read, multifunctional devises I found this Timex the best of all the other brands that I have tried in this category. Years ago, President Bill Clinton, who jogged regularly, was heavily chastised for wearing a very functional Timex Ironman watch in the Oval Office instead of what the professional politicians considered appropriate for a President. It was then that I realized how important it was for a person to wear a watch that they loved, and that was functional for them. At that point in time I had many other watches that I didn't jettison until later when I realized that I need only wear the watch that I loved and was useful to me. I still have another watch that I saved for "appropriate" times out but have only worn it once. The rest of the time my Timex Ironman stays on my wrist wherever I go.
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Reviewed in the United States on September 13, 2025