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Description
Human EPB42 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 an Erythrocyte Membrane Protein Band 4.2 (EPB42) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Erythrocyte Membrane Protein Band 4.2 (EPB42) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Erythrocyte Membrane Protein Band 4.2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Erythrocyte membrane protein band 4.2, also known as EPB42, is a protein encoded by the EPB42 gene. It is part of the erythrocyte cytoskeleton. It is an ATP-binding protein that regulates the binding of band 3 to acetylcholine. It may play a role in regulating erythrocyte shape and mechanical properties. Mutations in this gene are associated with recessive spherocytosis and recessive hereditary hemolytic anemia. | |||||||||||||||||||||||||||||||||
| 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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4.4 ★★★★★
Based on 14 reviews
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Product Reviews
★★★★★ 5
Durable
This is the best dog toy I have ever bought! I got my 1st one in 2013 and it still squeaks. Admittedly, there is only one squeaker left but it still works. My dog has played with this silly snake more than any other toy she has. My friend often brings her dog when she visits and then the 2 dogs play tug-of-war with the snake. These are small dogs;, one is a pug and one is a small mixed breed that weighs about 15 pounds. They pull and tug with all their might. Even so, my first snake has no rips or tears. The only thing wrong is that most of the squeakers have given up the ghost. That's why I decided to replace snake. I don't think my pug noticed the difference. I washed it as soon as it came out of the package just to make sure there was no residual die in the fabric as I knew my dog would have it in her mouth ASAP. I don't put my dog toys in the dryer but they just have to be washed from time to time. So, all is well with snake except that he is much louder now that all of his parts work. That's the other thing, don't buy this toy if you can't stand the noise.
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Reviewed in the United States on October 15, 2016
★★★★★ 5
Dog LOVES this toy
My dog, a Husky/Carolina dog mix, LOVES squeaky toys. Probably more than me, to be honest. Any squeak sound he hears throws him into "I must destroy it" mode. So, for Christmas, I purchased him this Outward Hound Kyjen Invincibles Plush Snake. He. Went. Insane. It was love at first bite. He was squeaking and chewing it all day. However, as I figured, this toy is not invincible. He's already shredded a good portion of it. I had to vacuum up pieces. Luckily, he's never been one to swallow pieces, but I have other dogs who might, so I have to keep a watch on it. If you have a dog who eats pieces, watch them carefully. Fabric can cause blockages in the stomach or intestines of the dog.
Overall, this was a great gift for my pup, and I recommend it to other humans with squeaky-toy-loving doggies.
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Reviewed in the United States on December 28, 2015
★★★★★ 5
Great toy
Great toy. My two German shepherds love it and it lasts a long time
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Reviewed in the United States on March 16, 2026
★★★★★ 4
Well Made Toy But My Standard Poodle Chewed Through it Anyway!
We have a standard poodle puppy that chews EVERYTHING! There isn't anything that she can't chew through - leashes, harnesses, toys - you name it and she tears it apart and if it's a toy, de-squeaks it and tears all the stuffing out. She has very powerful jaws! Now I have a couple of other Outward Hound toys that are made only of firehose material and those are still intact. But she loves the feel of the plush toys so I thought this seemed very durable and the inner part was of their Dura-Tuff liner, so I was hoping this would be a win win situation! The toy came and it IS well made and the outside plush is soft and inviting for the dog. Squeakers in every segment is a huge bonus with my dog as she loves squeakers. And no stuffing. Those nice qualities aside, after several weeks, she did manage to rip it apart, shred the material and de-squeak it! For an average dog or puppy, this might have worked wonderfully and lasted the entire lifetime. I still think its a quality made toy, but it just didn't last for my girl!! If this review has helped you in any way, please check yes at the bottom of the review.
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Reviewed in the United States on July 8, 2016
★★★★★ 5
The joy my dog gets from these toys is priceless!
I have a 5 year old Rottweiler, Poppy, who will absolutely annihilate any toy within minutes. I’ve had them all… same outcome. Stuffing all over within seconds and the enjoyment is short lived.
I love these toys. There are several squeakers in them so once she gets ones out, she still loves the toy because she can find another squeaker. Also, because it’s long (yet substantially sized) she LOVES throwing them around and whipping them back and forth - something she does even after all the squeakers are gone. That alone makes this toy worth it. She typically no longer plays with toys once the squeaker or stuffing are removed but she will continue to whip these snake toys around and they’re durable. I don’t have to worry about her eating small pieces of cloth that is ripping off the toy.
Having said all of that…. I’ve ordered her a few over the last several months and found that these toys are her favorite above all else. I’ve also found them to be a good value for me.
But… if I could give these toys 25 stars, I would. My Poppy girl was recently diagnosed with hemangiosarcoma and will have about 6 more precious weeks with us, if we’re lucky. So, I ordered her two more. Because, she can have all the toys, right? And the pure amount of joy that lights up her beautiful face every-time she gets a new snake is priceless. I will absolutely continue to order them to make her remaining weeks as delightful as possible.
I will be forever grateful for the happiness these specific snake toys bring my baby girl and could not recommend them enough.
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Reviewed in the United States on April 30, 2025