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Description
Rat N-MID-OT 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: 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: 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. 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. 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. 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. 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. 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. Preparation of 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 4000pg/mL). Then dilute to the following concentrations: 4000pg/mL, 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, and 0pg/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 4000pg/mL standard working solution into the first EP tube and mix thoroughly to make a 2000pg/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube is used 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 a capture antibody against N-terminal mid-segment osteocalcin (N-MID-OT). 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 N-terminal mid-segment osteocalcin (N-MID-OT) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat N-terminal mid-segment osteocalcin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Osteocalcin, also known as bone R-hydroxyglutamic acid protein (GLa protein), is a 49-amino acid protein. It is a non-collagenous acidic glycoprotein and a vitamin K-dependent calcium-binding protein. It is primarily synthesized by osteoblasts and odontoblasts, with some synthesis by proliferating chondrocytes. In its uncarboxylated form, osteocalcin acts as a hormone in the body, sending signals to the pancreas, fat, muscle, testicles, and brain, playing a key role in regulating bone calcium metabolism. | |||||||||||||||||||||||||||||||||
| 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 | 62.5-4000pg/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.8 ★★★★★
Based on 12 reviews
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Product Reviews
★★★★★ 5
Lots of slots to power devices all at once
Style: 7-in-1 USB-C Hub
Well made, good price, outlets precision sized so cords don't droop or fall out. Overall about as a compact as it could be. Enough power to plug into iPad and power phone, hearing aid charging box , and AA battery 4 slot charger.
Worth paying a little more for more slots.
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Reviewed in the United States on June 9, 2026
★★★★★ 1
Be aware of bad units being sold right now.
Style: 4-in-1 USB-C Hub
These USED to be a 5 star product from me, I've lost count how many I have purchased and included built into my products I manufacture. All of 2025 NO ISSUES at all, battle tested in the photo event industry buy some very large companies I have as clients. Come 2026 and the last two batch orders I have purchased I have had to replace 10 of them within a 2 month window, all for different customers because they stopped working in a short amount of time. Something happened in the Belkin manufacturing of these hubs in the past 3 months and I have lost my confidence in the stock that is available right now, I have moved to another HUB manufacturer for my products. I even tried calling Belkin quietly to make them aware of this issue and could not get anyone who cared that they had bad units being sold. So now it's to be very public about this issue and to let people know to be aware there ARE bad units in circulation of these hubs. ALWAYS have a backup on hand just in case! Sorry Belkin, you need to have a better process to receive feedback from other manufacturers that use your products in theirs. I hope I can regain confidence again someday.
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Reviewed in the United States on April 9, 2026
★★★★★ 4
Nice portable USB hub.
Style: 4-in-1 USB-C Hub
Nice little hub - output is 40 w though so a slow charger if using it to charge a phone off of a computer.
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Reviewed in the United States on March 17, 2026
★★★★★ 5
This hubs got what I need and drives seem to mount right away.
Style: 5-in-1 USB-C Hub
Might be a little early to write this review. But, I feel like I keep buying these types of hubs and they never seem to have the right configuration for what I need. Two USB-Cs, Two USBs and an HDMI. Appreciate that the ports are all on the same side so I don't have a spider web of cables going in every direction. They all fit rather snugly. MOST IMPORTANTLY, every external drive and flash drive seems to mount right away.
I have had major frustrations over the years with external drives not mounting or taking forever to mount. I'm sure most of this is due to the failing drives themselves, but I have a sneaking suspicion the hubs are playing a role too, but I don't have the electrical engineering degree to prove it.
Anyway, so far so good with this one.
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Reviewed in the United States on June 4, 2026
★★★★★ 5
Work as expected
Style: 4-in-1 USB-C Hub
Work as expected. Plug and play. For a USB-C hub, worth the price. Had no compatibility issues with my PC and a laptop. Haven't noticed any heat issues.
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Reviewed in the United States on May 8, 2026