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Description
Human PHB 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 and centrifuge 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. Cell Supernatant: 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., 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 the 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. Preparation before testing: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the gradient working solution of the standard: 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. 3. 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 as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 4. 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. 5. 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. 6. 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 a prohibitin (PHB) 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 prohibitin (PHB) content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Prohibitin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Prohibitin, also known as PHB, is a protein encoded by the PHB gene. PHB genes have also been described in animals, fungi, plants, and unicellular eukaryotes. Prohibitins are divided into two classes, designated type I and type II, based on their similarity to yeast PHB1 and PHB2. Every organism possesses at least one copy of a single type of prohibitin gene. Prohibitins are evolutionarily conserved and ubiquitously expressed. The prohibitin gene, located on the BRCA1 chromosome at 17q21, was initially thought to be a negative regulator of cell proliferation and a tumor suppressor. This antiproliferative activity was later attributed to the 3' UTR of the PHB gene, rather than the actual protein. Mutations in PHB have been associated with sporadic breast cancer. However, overexpression of PHB is associated with decreased androgen receptor activity and reduced PSA gene expression, leading to reduced growth of androgen-dependent prostate cells. Levels of the longer transcript are higher in proliferating tissues and cells, suggesting that this longer 3' untranslated region may function as a trans-acting regulatory RNA. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, cell supernatant, tissue homogenate, etc. |
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4.1 ★★★★★
Based on 560 reviews
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Product Reviews
★★★★★ 4
I recommend using the KONG Extreme Dog Toy
Style: Sport, Size: Large
Helpful for throwing as much as my dog wants to fetch. I actually had to get a cortisone injection in my shoulder a few months after getting my dog - never had a dog with this much energy before! The Chuckit really helps.
Downside: The ball that comes with it is larger than a tennis ball, so may be too much for smaller doge. I can't imagine my sister-in-law's french bulldog carrying it! Also, in our house it gets destroyed within minutes, as my dog is a strong chewer. For this situation, I recommend using the KONG Extreme Dog Toy, Black, size Large.
This is shaped sort of like a snowman, which means it bounces unpredictably at times. My dog eventually chews the "head" off of this toy, but the remaining 2/3 still fits in the Chuckit (and is actually easier to throw with the "head" missing!). One caveat: the KONG is significantly heavier than the ball included with the Chuckit, so be aware of that...
Also, we ordered our first Chickit in October 2015, then needed to replace it in May of 2017, as it broke. I think the plastic became brittle over that time, as we left it out in the sun & rain in Florida. Since it is cheap to replace, that's not a big deal. (We ordered three Kong toys over that same period of time!)
To summarize, it's a very helpful tool to get the ball further, faster, with less strain, than without it. Knowing there are alternatives to the included ball for heavy chewers makes it worthwhile.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on July 19, 2017
★★★★★ 5
Great Ball Launcher!
Style: Sport, Size: Large
Our dogs love this ball launcher! I can throw the ball long distances without any arm strain. The balls wear out, and as described by others, tennis balls don’t fit. Buy extra balls if this is of concern. We bought a few of the “Chuck It” rubber 3” balls and they have worked well for our dogs. It’s a great value and fun for the dogs.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on March 28, 2025
★★★★★ 5
Chuckit!! Just Chuck it!!
Style: Sport, Size: Large
Chuck it further than your skinny little arms can chuck it. Doggo loved this, I loved this and when we were done Doggo promptly hid it under the porch so I couldn’t take it from him. It’s not a squeaky toy, it’s for fetch. You wing it out toward the yard and it flies, bounces if it’s lucky then is attacked in an adoring manner. We like it!!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 31, 2026
★★★★★ 5
Great to exercise the dogs!
Style: Sport, Size: Large
Great to throw 3” KONG Ball (RED) with hole thru it! The thrower is the best to exercise dogs with retrieving balls!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 6, 2026
★★★★★ 5
The perfect toy for dogs!
Style: The Duo
This is perfect when you have 2 dogs who love to play Fetch! They no longer have to chase after the same ball! The balls that come with it are very sturdy, and the launcher sends both balls far enough away so that both dogs get plenty of exercise. It’s definitely worth the price!
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on May 26, 2026
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