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Description
Human CLCA1 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 conjugate are sequentially added to microwells pre-coated with Chloride Channel Accessory 1 (CLCA1) 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 Chloride Channel Accessory 1 (CLCA1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Chloride Channel Accessory 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Chloride channel accessory protein 1, also known as CLCA1, is a protein encoded by the CLCA1 gene. This gene encodes a member of the calcium-sensitive chloride conductance protein family. To date, all members of this gene family map to the same region on chromosomes 1p31-p22 and share a high degree of homology in size, sequence, and predicted structure, but their tissue distribution differs significantly. The encoded protein is expressed as a precursor protein that is processed into two cell surface-associated subunits, although the precise site of cleavage has not been determined. The encoded protein may be involved in mediating calcium-activated chloride conductance in the intestine. Protein structure prediction methods suggest that the N-terminal region of this protein is a zinc metalloprotease. | |||||||||||||||||||||||||||||||||
| 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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4.5 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 5
Training pad holder literally rises above the competition
Size: 24"X24"
We have been through a variety of potty pad products during our housetraining journey with our Havanese pup. The Petphabet training pad holder has been the best by far and has ended our search for a number of reasons:
Pros:
1. Grid design keeps puppy from chewing/tearing/destroying/eating the potty pad.
2. Size is perfect for our pup even as he is getting bigger (15 lbs) - mileage may vary for larger pups/dogs.
3. HERE IS THE REASON THIS PAD HOLDER STANDS OUT AMONG THE COMPETITION - The top grid is elevated off the surface layer where the pad lays, which allows an air gap between the standing surface and the potty pad. This allows the urine to fall to the pad without pooling and without your pup stepping in it and tracking it everywhere!
Cons/Areas for improvement:
1. We like blue but it's not for everyone!
2. The "posts" holding the mesh up from the base carry all of the weight of the dog and in some cases jumping or playing on the pad holder can cause the edges of the posts to cut through the potty pad liner, causing a (minor) leak. I would recommend the manufacturer design any subsequent models with larger, flat surfaces on the post supports.
3. The 4 piece design means the base is not solid but has a split down the middle that is not waterproof. This can be remedied by caulking the 2 sides of the base together with clear silicone assuming you will no longer need to disassemble the unit.
4. Poo can get caught in the grid if your pup has loose stool. If not removed quickly your pup may step on it and really pack it in there.
After many trials with other products, we are very happy with this training pad holder and have bought a total of 3 units for different areas of our house. I highly recommend for small dogs/pups!
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Reviewed in the United States on August 28, 2023
★★★★★ 5
Works Great!!!
Size: 18"X24"
I have 2 pugs and have trained them to "go" on a grid. I started with another brand (Klean Paws) but found this brand (Petphabet Puppy Training Pad Holder) to be superior in several ways. This training pad holder is designed much better. The plastic grid is well supported so the puppy does not get wet paws when waling on the grid. The overall design of the training pad holder makes it very easy to replace training pads. Standard training pads (17.5" x 23.5") fit the medium Petaphet puppy training pad holder perfectly. Just lay the pads flat on the bottom, place the grid on top and clip it all into place with the frame. The frame holds everything in place and keeps any loose ends, hidden from mischievous puppies.
It takes a lot of effort to train puppies to "go" on a grid, but it's worth it. I started with one grid in a crate. Then, I gaited off my kitchen area, with 4 medium grids (as shown in the video). You cannot expect a puppy or dog to just go to one spot and do their thing. Dogs like to sniff and walk around before they "go." They need room. I suggest 2 medium girds per medium-sized dog. I have 2 pugs and 4 grids. I reward them with a pinch of peanut butter, whenever I see them go on the grid. I simply pickup the #2 with TP and flush it down the toilet. I replace the pads, in the 4 grids, once a day.
NOTE: The sellers of the dog training pad holders seem to come and go. If you cannot find this particular training pad holder, do a search for Mesh training pad holder. Be sure to get a training pad holder that has 3 parts -- the bottom tray, the mesh grid, and a frame that holds it all together. Some of the simpler grids have just 2 parts (bottom tray and top). They are simpler, but do NOT keep paws dry.
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Reviewed in the United States on October 1, 2018
★★★★★ 4
Good quality, but my dog didn’t like it
Size: 24"X24"
The product arrived in perfect condition and looks well made. It’s sturdy and easy to clean, so I can see it working well for many dogs. However, my Cavapoo (17 lbs, 1 year old) never liked it. I tried for several weeks, but there was no way to get him to use it. I think it depends on the dog’s preference. For us, it didn’t work, but the product itself is good quality.
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Reviewed in the United States on August 20, 2025
★★★★★ 5
Excellent pee pad tray has "legs" to keep feet off of the pad
Size: 18"X24", Size: 18"X24"
My 10 week old Pekingese learned quickly to use it, and Pekingese are a very stubborn breed. We started training her by removing the grid because she was used to paper. It snaps out easily. We used pee pads with sticky tabs to attach the pad, then snapped the "frame" in place without the grid. Within a few days, she had figured it out, so we snapped the grid back in so her feet never touch the used pad. No more wet feet. I bought the medium size that is aqua blue.
For those that ask if it will fit their crate...measure your crate! My large crate is 42" x 23" and the tray fits two ways as seen in the pictures. My small crate is 23" x17" and the tray does NOT fit. I wish the company made a 10" x 14" tray for traveling. (pink???)
Start using it when you have a couple of days to give your puppy 100% of your attention. 100% attention!!
If she/he does not use it, then it is your fault for not paying attention. My puppy would go every 15 minutes at first! Put a WHITE plastic shower curtain liner on your floor so you can see if she misses it. The liner wipes off easily with Clorox wipes or goes in the washing machine. For those puppies who stand on the edge of the tray, but still miss it because their back legs are barely on it, help them walk forward and put plastic under the tray to catch any misses.
Now, at 4 months old, she seldom uses it, but it is available. Her pen is getting larger and larger. After she pees outside, we let her run around in in the living room for about an hour, then she goes back in the pen. Her attention span is short and she can't always make it back to her tray in the pen. My husband always brings her in the house after she has peed one time outside, but if I take her outside, I wait until she goes at least 3 times and there won't be accidents in the house. Sometimes, it takes more than 1 time to empty their bladder.
This pad tray is not like others on the market. Look closely at other grids, and you will see that other trays do not have the "legs" on the under side of the grid that keep the plastic grid from touching the used pad. My puppy tried to chew on the plastic tray, but she is always in our vision so we just say, "Ahhh!" and she leaves it alone. No problems with chewing. The Ahhh!" stops her every time, and we say it to stop her from doing anything wrong. Some people say their dog chews on it. Just like children, dogs need to be taught. Don't complain about how the pee pad or pee tray doesn't work. Teach your dog or your dog to not chew on it.
Like others have mentioned, my puppy likes sleeping on the tray at night, because it has air circulating under it, so it is cooler than her bed. (Not the fault of the product!) We can't break her of that habit without confusing her or removing the tray. She may stop using it if we teach her to get her off it. I have been putting frozen gel compresses wrapped in a towel in her play pen during the day, and as long as the compress stays cold, she will lay on it instead of the pad tray. Always watch, so a dog doesn't chew on the gel compresses.
I don't know why some people have said that urine runs out, because it is sealed. Maybe they are not changing the pads often enough. I started out changing them twice a day, then since my puppy started flea and tick medicine, she could start going outside. I changed the pad once a day then. Now, at 4 months old, she goes outside, but we put it in her large kennel at night. (The tray is too big to fit in her small dog kennel.) For the last three days and nights, she has not used it at all, but it is there just in case.
If your tray does not have rubber pads on the bottom, buy some or put the tray on a piece of rubber kitchen shelf liner. My tray doesn't slide. If your dog runs to it and jumps on quickly, it may slide, so put something under it.
Remember, your dog may not want to walk on it, but it is up to you to teach the dog to walk on it. They don't get a choice! You are in charge!
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Reviewed in the United States on September 17, 2020
★★★★★ 5
Hands down, best pee pad holder
Size: 18"X24"
I did so much research to find the right puppy pad holder. I would give this 10
stars if I could.
It’s softer plastic but not flimsy. The holes are comfortable for puppy feet. I see a lot
with metal grates and pictures of injured doggy feet. Our pups are small and they walk on it just fine. I used to go through 10 pee pads a day. Now one pad lasts all day. Pups can’t go wee wee and trail it all over the house. Since it’s the same pee pad, pups look for the smell and are motivated to pee pee in the same spot. Puppies used to wake up several times a night because they would pee, step in it, tear up the pee pad, make a mess. The night time drama has definitely decreased significantly.
Easy to lock, puppies can’t get into it. Easy to open to change out the pee pad. Comfortable to clean in a larger kitchen sink. Lightweight. Sometimes the doodie will go through the grates and sometimes it sits right on top, depending on the size of the doodie. Cleaning doodie stuck in the grate is never fun, but I use an old toothbrush and it works just fine.
Very happy with this item.
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Reviewed in the United States on January 29, 2026
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