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Description
Rat FLOT1 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 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 uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate are sequentially added to microwells pre-coated with a flotillin-1 (FLOT1) 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 flotillin-1 (FLOT1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Flotillin-1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Lipid raft signature protein 1 (FLOT1) is a protein encoded by the FLOT1 gene. It encodes an integral membrane protein associated with caveolin. FLOT1 has been shown to interact with SORBS1. Diseases associated with FLOT1 include Mycobacterium marinum and cortical dysplasia complex, among other brain malformations. Pathways associated with FLOT1 include cytoskeletal signaling and programmed cell death. Gene Ontology (GO) annotations associated with this gene include protease binding and ionotropic glutamate receptor binding. An important homologue of this gene is FLOT2. | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
Based on 16 reviews
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Product Reviews
★★★★★ 5
My dogs favorite balls
Size: Medium
These are great for my dog who always has to have a ball in her mouth. Not too hard so I don't have to worry about her teeth. They last for ever. They do lose their shape a little over time (like I said, she always has a ball in her mouth) they become a little oval in shape. They still bounce, she still chases it and she still chews on it so I don't think she notices or cares. I've gotten her a bag full of these over time and I have only seen one that she chewed a chunk out.
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Reviewed in the United States on September 28, 2023
★★★★★ 5
My Dachshunds Love These
Size: Small
These balls are durable, flexible material, highly chewable without being easily destroyed. I stuff them with Milkbone Maro Snacks, which are just the right size to twist into the holes in the ball. My dogs literally get an hour+ chewing and chasing entertainment from these. They chew them, they sometimes 'pop' out of their mouths, go rolling across the floor, dogs chasing them. This satisfies their urge to hunt prey. Dachshunds can easily tear this kind of stuff-it-toy apart. These have lasted for a long time without tearing or shredding. I would NOT recommend them for a dog much larger than a tweenie weenie. They're a bit on the small side and might be swallowed by a larger dog. It would be cool it they also came in a larger size (and probably ball wall thickness also) for the bigger dogs to enjoy. These stuff-it chew balls are better than the kong balls my dogs had before these. Highly recommend them.
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Reviewed in the United States on April 19, 2024
★★★★★ 5
My puppy’s favorite fetch toy!
Size: Small
Update Aug 27, 2024: After 10 months, these are still his favorite ball. He’s obsessed with fetching and plays with them all day. They do bounce really high. Unfortunately, the small size aren’t available for sale anywhere anymore. I tried the medium size, and he has a bit of trouble hanging on to them, but he manages. Weirdly, he prefers the orange one, but he also plays with the blue one.
My 7 month old Shih Tzu puppy loves to fetch, and these are his new favorites. They’re small enough to fit in his mouth, and they are made of hard plastic, so he’s able to hold on to them. They have a lot of bounce, and this adds to the fun for him. I highly recommend them.
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Reviewed in the United States on December 17, 2023
★★★★★ 4
Genuinely bummed that these great balls can’t stand up against my Chloè.
Size: Medium
I love all the Chuckit brand of balls. I found that the Strabo and glow balls in the Chuckit line are the softest, and as such, don't last as long as the rest of Chuckit line of balls. Having said that, they do last longer than than every other brand I've tried (and I do believe I’ve tried every brand available on the market today.)
The strato balls do bounce higher than any of the other balls in the Chuckit line. They really are a good value.
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Reviewed in the United States on March 30, 2022
★★★★★ 5
Great dog balls at a great price
Size: Medium
The balls are a favorite for my dog. I could have done without the hole through the middle because I don't do treats in balls anyway. My dog loves balls for their own merit. I needed replacement balls for my Nerf Dog Ball launcher. It seems the originals have disappeared from the market. I took a chance because the price is right and this Chuck-it ball works very well so we're liking this ball a lot. So far my Dachsund, who can destroy a tennis ball (the kind they are selling as Nerf Dog replacements) in 3 minutes has not done any damage to this one so that's a win. I recommend this as a much better ball than the fuzzy tennis balls. I like the colors too since many dog toys are green (which gets lost in the lawn, etc.) or red (even though dogs have red/green color blindness. Blue and orange are good and pretty easy to find. I liked these well enough that this set was my second purchase so I will have spares in case the others are lost.
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Reviewed in the United States on May 21, 2023