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Description
Rat OCLN 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 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 an occludin (OCLN) 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 occludin (OCLN) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Occludin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | OCLN, also known as claudin, is an enzyme encoded by the OCLN gene. It oxidizes NADH. It was first identified in epithelial cells as a 65 kDa integral plasma membrane protein localized at tight junctions. Along with claudins and zonula occludens-1 (ZO-1), it is considered a major component of tight junctions. It is not only present in epithelial/endothelial cells but is also abundantly expressed in metabolically active cells lacking tight junctions: pericytes, neurons and astrocytes, oligodendrocytes, dendritic cells, monocytes/macrophages, lymphocytes, and myocardium. It influences key aspects of cellular metabolism, such as glucose uptake, ATP production, and gene expression. It plays a key role in maintaining the barrier properties of tight junctions. Consequently, its mutation or deletion increases epithelial leakiness, a crucial barrier to cancer metastasis. It also plays an important role in apoptosis. Mutations in OCLN are thought to be the cause of zonular calcifications with simple convolutions and polymicrocrystals (BLC-PMG), an autosomal recessive neurological disorder. | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
Based on 29 reviews
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Product Reviews
★★★★★ 5
Great toy
Color: Grey
My girl loves this toy. She has a lot of toys but this is her favorite. She is a 26 lb mini golden and we have had it over a year. It is just now starting to show wear. Once it is completely worn out I will buy it again!
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Reviewed in the United States on February 20, 2025
★★★★★ 5
Durable product.
Color: Grey
My dog loves it. Very durable.
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Reviewed in the United States on April 12, 2026
★★★★★ 1
15 Minutes….with update x2
Color: Orange
Within 15 minutes my 10-month old puppy had one of the moose antlers torn half off. I’ll see if he is able to destruct any more of this “indestructible” toy. If it holds up I’ll come back and upgrade the number of stars but for now…not impressed.
UPDATE:
Sadly he was able to rip it open via the appendages (the legs). The main body of the toy seems pretty tough but no dog is going to keep away from the legs. Downgrade to 1 star.
UPDATE #2:
I just received my second attempt at using this “indestructible” toy. This time I went with the rino since the legs are rope and not a sewn on leg. Well, in about 5 minutes my puppy had the horn torn and the horn provides direct access to the inside stuffing. As mentioned above, the legs are rope. The design was pretty smart in that both the front legs is one posted of rope going through a hole made in the stuffed toy. Same for the rear legs. This prevents the dog tearing them off and getting access to the stuffing. Sadly the horn being torn off right away makes this toy destructible.
I think the only way they may be successful calling this an indestructible toy would be if they made a ball out of the body material or perhaps something in the shape of a bone.
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Reviewed in the United States on October 9, 2023
★★★★★ 4
AWESOME TOY for aggressive chewers
My 4 legged fur baby has only had for a few days. Now we’ve purchased several others in the past and he LOVES them. EXTREMELY durable. He is a dachshund and loves to destroy his toys. It’s almost like it’s a mission to see how fast to get the stuffing and squeakers out of his toys and can say he has been unable to destroy any. Now the reason I gave this only 4 stars is because the end of the tail has hair like material and he did start tearing this apart. Was finding the hair on his blanket and hanging out of his mouth. So I just trimmed the tail and he is back to loving on his toy. I would highly recommend this to anyone.
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Reviewed in the United States on May 2, 2021
★★★★★ 5
Cute and my dog loves it
My dog loves toys she can play rough with both her teeth and paws and this one was loved the second it came out of the packaging. We liked that the eyes are sewn patches so nothing to swallow if it detaches (other than the material). The knotted ropes that form it’s arms and legs actually move through the body from side to side, which makes tugging games fun and interesting. We also liked that there were no dangling easy-to-chew off bits, for example, the ears are tiny nubs so she can’t get them off with her teeth easily. The stuffing is also firm and full so it’s like a soft toy to cuddle up with but with rope limbs to sink her teeth into. She rates it highly.
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Reviewed in the United States on July 9, 2021
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