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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 1229 reviews
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Product Reviews
★★★★★ 5
Perfect puppy starter kit and gift! Perfect for plane rides and kennel!
Color: Neutral, Color: Neutral
Perfect puppy gift! We use the kennel as her safe haven, and for potty training . This heartbeat tool is amazing and helped our puppy get acclimated to her kennel. She goes into her kennel and sleeps with it on her own. It calmed her down on the plane completely. We had a 6-hour flight and she did fine with the heartbeat toy. At home innthe kennel, the whining was very very minimal the first couple days, then it stopped. As you would hear and see her snuggle up and fall asleep. I think it is a bit pricy, but not too bad for the benefit. The heartbeat is seperate and is putninside a pouch on the stuffed animal. The blanket is soft, beautiful and perfect size. Good quality fabric. Both chew toys were loved by her.I do notice my large breed puppy prefers the rope and small tennis ball type chew toys. However, she plays with these too. Haven't used the heating packs yet, they go inside the pouch with the heartbeat item. I am sure we will because my puppy does love to be in front of my small heater under my desk to keep my feet warm. The packaging was extremely nice and fancy and was complimentary to the items inside.
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Reviewed in the United States on January 10, 2021
★★★★★ 5
Best Idea for Puppy's First Day Home
Color: Pink
I was a bit reserved on purchasing this kit because it was a bit expensive. After a month now with my pup at home, this was the best investment for us and our GSD. On the first night, we used the heart beating snuggle puppy and blanket. She slept the entire night without an accident or a cry. The heartbeat in the snuggle puppy and the heat pad was the best idea. Our puppy never felt alone (just fyi, her crate was in the room with us for the 2 weeks).
A month later, she is so protective of her snuggle puppy. We only used 1 heat pack to soothe her during the first week. The heat packs last a while. Our GSD now uses the snuggle puppy as a pillow or an object to be close to while she is in her crate. Any new puppy owner could definitely benefit from using this. Of course, with all things puppy, it does require a bit of time to correlate toys as a positive thing. We exclusively keep the snuggle puppy in her crate and make it a part of her secure place.
My GSD puppy did really care for the teething heart toy. Even after freezing it, she didn't care for it. She also outgrew the small toy. She will chew it from time to time but it doesn't entertain her much.
I found the kit, overall, very helpful with our puppy's adjustment to our home. She sleeps through the night since 8 weeks old and now at 12 weeks old. We've never had an accident in her crate overnight with the help with the snuggle puppy.
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Reviewed in the United States on April 5, 2020
★★★★★ 5
Amazing kit for your puppy
Color: Blue
We’ve had 6 puppies over the years, and this is the first time we’ve had this wonderful little kit to welcome our little one home. We had the blanket with his litter so it smelled like them when he came home. He he loves that blanket, he loves the dog with the little heartbeat, and the teething toys.
The 5 star review is because everything in this kit is so well made, really wonderful quality, and a great value. The blanket is a very nice thick plush, not some cheap one. The stuffed dog is big and fluffy and well made, the heartbeat works wonderfully, and the little chew toys safe and well made.i would encourage anyone who’s bringing home.
A little one please get this first. It’s going to make your pup more comfortable when they get home, and will help then sleep better during the night and their naps. Very Well Done !
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Reviewed in the United States on June 20, 2021
★★★★★ 5
A must have for a new puppy.
Color: Pink
I just got my first new puppy in many years, and I was searching for ideas of what items I should get to be ready for her. A dog forum I belong to gave suggestions for items to prepare for a new pup. One of the items listed was a snuggle puppy. I was not familiar with the product, and did some research. I had always heard that lonesome puppies respond well to a hot water bottle and ticking clock for comfort. A friend had suggested a stuffed animal. I found all of that in one package - The Snuggle Puppy! And even better was the complete new puppy package. The blanket is adorable, very soft and the perfect size. The toys are perfect, and they include extra warmers for the puppy. I keep my girl in a kennel at night with her blanket and puppy. The first night I tried just the blanket, and she cried and howled for 15 minutes. Then I turned the heart beat on and put the puppy in the crate with her. Within 5 minutes she was quiet and stayed that way all night. I have used it every night since and not a peep. (It's been a week now). I would highly recommend these products to anyone bringing a pup home - it was a sleep saver for sure and she seems to love it.
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Reviewed in the United States on February 3, 2022
★★★★★ 5
Great for a small puppy
Color: Pink
Got these for my puppy, a German shepherd, while it was functional, she loved it, as it was chewability, durability and ease of use.
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Reviewed in the United States on November 7, 2025