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Description
Human PIAS3 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 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 the E3 SUMO-protein ligase PIAS3 (PIAS3). 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 E3 SUMO-protein ligase PIAS3 (PIAS3) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human E3 SUMO-protein ligase PIAS3 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Protein inhibitor of activated STATs 3 (PIAS3), also known as the E3 SUMO-protein ligase PIAS3, is an enzyme encoded by the PIAS3 gene. PIAS3 was initially discovered to interact specifically with phosphorylated STAT3 in interleukin-6 (IL-6)-activated myeloblast M1 cells. This interaction is mediated by PIAS3 binding to the STAT3 DNA-binding domain. Consequently, STAT3 transcriptional activity is inhibited by physically preventing its binding to target genes. Subsequently, it was also discovered to be a regulatory protein of other key transcription factors, including MITF, NFκB, SMADs, and the estrogen receptor. It also functions as a SUMO (small ubiquitin-like modifier)-E3 ligase, catalyzing the covalent attachment of SUMO proteins to specific target substrates. It directly binds to several transcription factors and either blocks or enhances their activity. Alternatively spliced transcript variants of this gene have been identified, but the full-length nature of some of these variants has not yet been determined. | |||||||||||||||||||||||||||||||||
| 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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4.1 ★★★★★
Based on 16 reviews
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Product Reviews
★★★★★ 5
Simple! Easy! All We Needed!!
Size: Large Triple
These were the only durable rubber like rings that were both the same material; not too thick (not heavy) so my 1 year old Pit, Dixie, was able to latch on to one ring while we grabbed the third ring, leaving enough space so she won't accidentally bite us when grabbing the rings to play. They were also the only ones that came interlocked and cannot be taken apart. She is also a big chewer, which means she can gnaw on these without tearing them apart. The rubber is thick enough to play, but thin enough to twist and flex with her. So far, they are sturdy. She was immediately so excited! Simple and easy!
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Reviewed in the United States on May 8, 2026
★★★★★ 5
Great durable dog toy!!
Size: Large Triple
We have a medium-sized dog (45 lb mixed Aussie/Hound) who is a pretty aggressive chewer. He also loves playing tug-of-war with his canine and human siblings using these rings. This is easily his favorite toy. We bought one a little over a year ago that he plays with daily. If finally broke this December just in time to buy a replacement for Christmas!
The prior one finally broke when the middle ring came apart at one of the thinner rubber points. He still plays with the intact outer rings as well as his new toy.
I wanted to dock this 1/2 star for a few reasons: 1) it is not completely invincible, although I would argue a toy that lasts over a year with pretty constant play for an $11 price tag is a great value. 2) the one we received is not yellow/black. It is blue/green. I was personally thrilled it was blue/green because it ended up being an exact replacement of the one we had previously. However the description does not say "colors vary" and it was hard for me to tell when ordering if I was ordering the correct replacement toy. A clearer description would be appreciated. 3) This is a great toy for one dog, but our other dobbie/cattle dog mix has no interest in them. So not all dogs will love this toy (obviously).
4.5 stars isn't an option so I'm giving this 5 stars because it's just about as close to a perfect dog toy as one can buy (at least for our dog).
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Reviewed in the United States on December 31, 2016
★★★★★ 4
Great toy, as long as dog is NOT ALLOWED to chew on it
Size: Large Triple
Let me start by saying that I like this dog toy. My big dog LOVES tugging with it. As long as you keep away when the dog is in a chewing mood, it lasts a long time (my 1st one lasted a year), however, it WILL NOT last if the dog chews on it, as I found out with this one. A friends dog chewed on it while she was behind a snow mound (mostly out of our sight) when it was only a week old. One end ring was barely holding together when we discovered her chewing it. Fortunately it was an end ring, so I still have 2 connected rings for tugging with.
It is a great tug toy! But it is also a TERRIBLE chew toy, so make sure to take it away when your dog starts to chew on it and it will give you long service.
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Reviewed in the United States on February 14, 2021
★★★★★ 5
Fabulous toy!!
Size: Large Triple
My GSD is always up for a good game of tug-o-war. I purchased these in large and I think I love them as much as my dog. They are super strong and hold up to his 100lb self pulling on one end and my “blah blah-lb” self pulling on the other. (Ha! You thought I would say how much I weigh) I even bought another pair for our cabin so we have some to play with at both places. He isn't a sit and chew on things kind of dog. He gets plenty of playtime and bully sticks for chewing. Not to mention that I wouldn't let him use them as a chew toy anyway. There are toys for play and toys for chew. Therefore, I cant say anything on how they hold up for chewers. So if you let your dog just sit and chew on it instead of giving them something designed for strong chewers, then no it might not hold up to that. All I can say is my German Shepherd is 100lbs with sharp teeth and stronger than me now and honestly he is probably stronger than I have ever been in my life, and they hold up to the pulling and tugging of both of us. Thats what they're designed for and they serve their purpose excellently.
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Reviewed in the United States on January 22, 2026
★★★★★ 1
Won't purchase this one again.
Size: Large Triple
J.W. used to make a ring toy such as this, that was softer & thicker with a smaller black ring in the middle, that our dogs all, absolutely loved!!!!! But this one totally bores them to the point none of the dogs will pay it any attention! Please bring back the original 3 ring toy, that J.W. has now changed to just the one ring! I feel like they must have discontinued the toy with the 3 rings they used to sell, with the smaller black ring in the middle? I won't waist my $ on another one of these! Sadly, none of our 3 dogs will play with this specific toy at all! & I generally prefer to purchase J.W. brand toys for our dogs. But this toy doesn't attract them in anyway. It just sits around.
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Reviewed in the United States on March 6, 2026