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Description
Human CALCOCO2 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 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 calcium-binding and coiled-coil domain-containing protein 2 (CALCOCO2) capture antibody. 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 calcium-binding and coiled-coil domain-containing protein 2 (CALCOCO2) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader, and the sample concentration is calculated. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Calcium-binding and coiled-coil domain-containing protein 2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Calcium-binding protein coiled-coil region 2 is a protein encoded by the CALCOCO2 gene. The protein encoded by this gene is a subunit of the nuclear domain 10 (ND10) body. ND10 bodies are nuclear domains that appear as ten small dots per nucleus on immunohistochemistry. Based on their resistance to nuclease digestion and salt extraction, they are thought to be associated with the nuclear matrix. ND10 proteins are removed from the nucleus following HSV-1 infection and may play a role in the viral life cycle. Diseases associated with CALCOCO2 include granulomatous disease, chronic, autosomal recessive, and salmonellosis. Gene ontology annotations associated with this gene include protein homodimer activity. An important homologue of this gene is TAX1BP1. | |||||||||||||||||||||||||||||||||
| 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 | 0.312-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.2 ★★★★★
Based on 893 reviews
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Product Reviews
★★★★★ 3
I’m a little disappointed.
Format: Kindle
I usually like Jillian West’s books but this one was missing a lot for me. The pregnancy didn’t come across as real. She’s on her feet for 12 hour days but is perfectly healthy at 8 months pregnant? Yet the week she moves in all of a sudden she’s not? She is planning on actually running during one of the plot buildups. But at 8 months pregnant that’s incredibly hard to do. The lack of breathing ability and lung space, the change in body center, mass, and gravity. All of it prohibits running, unless you’re an athlete this didn’t come off as at all realistic.
I didn’t feel any connection with the alphas. There wasn’t any emotional connection. It could be because of the tense it was written in. But I didn’t get any deep feelings out of this. It came across as checking off boxes. Even the spicy scenes weren’t really believable for me. I wanted to see them fall for her, and it just kind of all fizzled. Even Bishop.
One thing I did really like was the ending. I did not see it coming and I’m interested in reading book two because of it. But on the whole this book was mostly disappointing for me.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 16, 2024
★★★★★ 4
4.25 stars
Format: Kindle
Vale is an 8 month pregnant omega working as a waitress at a strip club and a cam girl. She starts to get very creepy vibes from a regular at the club, and her baby daddy ghosted her. She has had an online relationship with a man named Bishop through her cam girl status. One night, bishop was paying to watch her sleep and ansthe creepy regular Andrew break in and watch her sleep he tells vale to come to him at his business now. She flees and finds herself at a large security company with some.hot of alphas who are there to help her. This imegaverse is a little different than I have read, but I am thoroughly enjoying it. Vale is not a traditional omega she was raised by a single beta mom, and the alphas are not normal alphas they have never really loved pack life. But they are ruthless mercenaries. They need her, and she needs them. I love the aspect of the stalker and now the plot twists at the end, so so good. Sometimes, it seemed a little slow and stale mated, but since this a duet, I think It was just her starting to have Vale get to know her alpha suitors. Cliffhanger for sure with this one.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on September 9, 2024
★★★★★ 5
That ending 😫
Format: Kindle
I fell into a false sense of security and really thought this was gearing towards a happy ending. Then I realized there’s no work they don’t punish Andrew. I really liked Vale’s character. I don’t normally read books with pregnancy but going into this knowing she was pregnant made it more enjoyable for me.
I loved Bishops devotion to her and her happiness. I also loved that Holt and Mercy couldn’t fight their attraction to her. I love scent matches so very much. I’m so curious to see how this duet will end up. And I need to pay more attention and notice that a book I’m starting is a duet to begin with lol
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 21, 2025
★★★★★ 5
oh wow
Format: Kindle
I just knew there was something about Cooper! I’m wondering if he’s about to be included but damn I’m glad he’s at least not a rapist and creepy guy, he just got called on assignment and had to go! This should be interesting! She’s gonna run and then what’s his face is gonna grab her. I’m worried! Wow that was a great book and cliffhanger! Loving this!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on December 27, 2025
★★★★★ 5
Wow.
Format: Kindle
Okay I’m usually not one for stand-alone’s I’m an avid series reader but my goodness am I so happy I read this! This story was brilliant & so absolutely mesmerizing. I loved reading about each character and their struggles as well as what helped them to move forward. The ending definitely brought tears to my eyes so hard. I truly wasn’t expecting some of what happened in this story. There is about to be a spoiler I am going to reveal so please stop reading if you don’t want the spoiler !!!! ⚠️ ⛔️ ‼️
I loved that the author didn’t do what most authors do with irredeemable male characters. I truly was hoping that Nate Jr. would be apart of the pack after the way he treated Astrea bc he truly didn’t deserve it. Though I must say you did a wonderful job or redeeming him as a person. I cried my eyes out when he walked into the story. I was truly terrified he was going to be a bad guy to the end. However you truly did him such a justice by having him realize his faults & having him redeem himself in the most wonderful way. I’m so sad that he didn’t get to hear how much his brother loved him & forgave him before dying. But again you wrote that ending so beautifully & I just can’t express how much I loved this story & how you took a different route than most authors I have read have. You are a remarkable author Cinder Blaze & I thank you generously for creating such a masterpiece.
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Reviewed in the United States on April 28, 2025
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