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Description
Rat P4HB 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. Centrifuge the specimen 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. 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 the tissue and mince it. 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 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. 4. 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 100 ng/mL). Then dilute to the following concentrations: 100 ng/mL, 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL, 1.5625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each tube. Pipette 500uL of the 100ng/mL standard working solution into the first EP tube and mix thoroughly to make a 50ng/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 utilizes 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 capture antibody against prolyl 4-hydroxylase subunit beta (P4HB). 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 prolyl 4-hydroxylase subunit beta (P4HB) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Prolyl 4-hydroxylase subunit beta ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Protein disulfide isomerase, also known as the β-subunit of prolyl 4-hydroxylase, is an enzyme encoded by the P4HB gene. Unlike other prolyl 4-hydroxylase family proteins, this protein is multifunctional and acts as an oxidoreductase for disulfide formation, cleavage, and isomerization. The activity of P4HB is tightly regulated. Both dimer dissociation and substrate binding likely enhance its enzymatic activity during catalysis. | |||||||||||||||||||||||||||||||||
| 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 | 1.56-100 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates and other biological fluids |
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4.6 ★★★★★
Based on 13 reviews
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Product Reviews
★★★★★ 5
Top Class Watch
Color: sliver band blue face-G5885G
I really love this watch, its very bright in the night, it was very easy to set the time on it, I also love the weight of this watch, it's definitely of top class quality, thankfully I didn't need the tool to adjust the band because it fit perfectly fine, plus the quartz system is working quite well, hasn't missed a second since I set it. Time keeping is amazing. Overall, I love this watch so much, I'm thinking of buying the black face one as I bought the blue face one.
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Reviewed in the United States on May 27, 2026
★★★★★ 5
Good bang for the buck
Color: Silver Men Watch/Black Dial-2870
Great looking cheap watch... So far working great and keeping time 👍
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Reviewed in the United States on May 26, 2026
★★★★★ 5
Tracking Process & Quality of Product
Color: Black Watches for Men/Black Dial-2870
I don't know where to review the tracking process so I will do it here. I just love the fact that they list stop by stop where your order is. I ordered this for my son for his graduation present and was very excited to give it to him. I was able to see how many stops the driver had before he got to me which allowed me to not only have a better idea of its arrival time, but I was also able to enjoy a buildup of excitement as it got closer and closer.
As for the quality of the product ~ both my son and I are very impressed and more than satisfied with the watch so far. I was very trepidatious as so many items, especially jewelry items, look stunning, gorgeous, beautiful, high quality in the pictures online and are nothing short of utterly disappointing when you get them. This watch renewed my belief that there are honest sellers out there. I will say that we just got the watch today so this is purely about the initial presentation and look of the watch.
So, the packaging was super nice, high-end quality. Each piece (bands, watchface) were separately wrapped in nice, thick plastic to protect from scratching. The boxes (box for watch & slip cover outer box) were great quality (you know, so many companie's packaging falls apart at barely a touch, which makes me wonder about the quality of what's inside). Thorough, grammatically correct (mostly) directions included; also had tools for taking out links along with fairly easy to follow pictured & written directions. We did have to use a separate pin with a head (like tailors use to mark hem points) to push out the pins bcz the one included bent.
One thing I really appreciate is that the company said they are there anytime and to please let them know of any issues. Usually companies do all they can to make it as hard as possible to be contacted.
I couldn't stop looking at the watch! It DEFINITELY looks SO MUCH MORE expensive than it was! And the workings move like a Rolex (smoothly), not jumpy like the workings of normal watches. Even my husband remarked on how "Rolexey" it looked. I ordered the all black watch.
Again, we've only just gotten it, but if the initial presentation is any indicator of its performance, WE'VE GOT A WINNER!!
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Reviewed in the United States on July 18, 2023
★★★★★ 4
Good looking cost effective
Color: Gold Silver Stainless Steel/Blue Dial-2870, Color: Gold Silver Stainless Steel/Blue Dial-2870
Good for the price. Looks nice from a distance. Feels heavy. Is a bit noisy when you walk. But value for money.
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Reviewed in the United States on May 3, 2026
★★★★★ 1
Low cost watch returned.
Color: Gold Silver Stainless Steel/Black Dial-2870
I returned this watch the next day after receiving it. It was very attractive and I hope it would work as advertised. The watch came in shrink wrapped plastic that was very tough to removed. I feared damaging the watch. I set it then tried some of its features. The stopwatch worked but it did not reset to the zero position properly. The 24 hour time indicator was off by one hour. The rotating bezel had black lacquer on it that was tender to the touch. A fingernail could have damaged it and a very small nick had already started out of the box. If everything had worked properly I would have kept it for the money spent. I guess you get what you pay for so I am now shopping for a higher quality watch.
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Reviewed in the United States on August 10, 2024