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Description
Mouse MK 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 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 utilizes 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 Midkine (MK) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of Midkine (MK) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Midkine ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Midkine (MK or MDK), also known as neurite outgrowth promoting factor 2 (NEGF2), is a protein encoded by the MDK gene. It is a low molecular weight, basic heparin-binding growth factor and forms a family with dystrophin (NEGF1, which has 46% homology to MK). It is a non-glycosylated protein composed of two domains held by disulfide bridges. It is the product of a developmentally important retinoic acid-responsive gene that is strongly induced during midgestation, hence the name midkine. It is primarily restricted to certain tissues in normal adults and is strongly induced during tumorigenesis, inflammation, and tissue repair. It has pleiotropic effects, including cell proliferation, cell migration, angiogenesis, and fibrinolysis. Its receptor is believed to be a molecular complex containing receptor tyrosine phosphatase zeta (PTPζ), low-density lipoprotein receptor-related protein (LRP1), anaplastic leukemia kinase (ALK), and mitochondria. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.8 ★★★★★
Based on 8 reviews
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Product Reviews
★★★★★ 5
Ends in a cliffhanger
Format: Kindle
Thankfully, the next book is already available.
Roxy Collins is the queen of omegaverse slow burn. This particular series is omegaverse with wolf shifters.
Thoroughly enjoyed this book! Lots of suspense and trying to figure out what is true and what's really going on. I loved it!
Pretty spicy once the action starts. Heats are pretty much always spicy. There is a little bit of male interaction, but not full-on shmex.
Definitely recommend.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 29, 2025
★★★★★ 5
Epic!!!!
Format: Kindle
Absolutely addicted from the first page. The twists and turns, world building and character development all come together for the perfect Omegaverse. And let's not forget that spice because knots are life and these are spectacular. Running for book #2 because that cliffhanger was torturous.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on August 17, 2024
★★★★★ 5
A good read
Format: Kindle
A good read, just fluffy cuteness, no antagonism. I like all the characters. It could have used another round of editing however, glanfds being one error that cracked me up, and my personal pet peeve was that the author kept using the word fill instead of feel, which I promise you are not interchangeable haha, but it's definitely better than the majority of books I read on here mistake-wise.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on November 28, 2024
★★★★★ 4
amazing
Format: Kindle
Knot the Bride was a fantastic read! The characters were all amazing and well-developed. It was easy to like them all. Sophia, Luca, Nick, and Gavin were all perfect for each other. It was such a charming story that had me hooked the entire time. I did wish there were POVs from Luca, Nick, and Gavin but it was still an amazing book without it.
I am excited to read the next book in the Willowside Omegaverse series! This is definitely a must-read for fans of omegaverse romance!.
I received a free copy of this book and am voluntarily leaving a review.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on April 25, 2025
★★★★★ 3
3 Star Read,
Format: Kindle
This book wasn't bad, but wasn't my cup of tea. It's highly disappointing because the storyline is so original. There is no real conflict or resolution, so the entire thing feels flat.
As a lover of omegaverse books, I know there is a ton of variety out there, and ov is really up to the author. But this one is weird. Omegas have multiple scent glands all over their bodies and go into week long heats every month. Alphas have knots in the middle of their shaft instead of the base, and the knot doesn't always swell, no explanation of when or why. It doesn't engage at all when the mouth is in play.
I also didn't enjoy the author's writing style. Each paragraph is only 1 or 2 sentences long, and the entire book reads very stacato. The conversations are stiff and unnatural feeling. Everything is very repetitive, both in word choice and in thought. The same thing is repeated 3 or 4 times over a single page, multiple times over. I ended up doing so much skimming.
The first 50% of the book is all slow burn, and the last 50% is almost straight mediocre spice. This wouldn't have been all bad if the grammar and spelling errors didn't start at the exact same time. Tongue is repeatedly misspelled in the middle of the spice.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 14, 2024
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