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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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4.7 ★★★★★
Based on 15 reviews
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Product Reviews
★★★★★ 5
Blood and Muscle
Format: Kindle
I had this on my shelf for a while. lG reviews were great, the cover was pretty, and it sounded really interesting.
It was better than I expected! Love when that happens🥰
The story was great, the writing was good, and the characters were amazing! Ava and Lou's chemistry at first was a little charged. But as the story went and their relationship developed, it turned into something more tender and very intimate. Throw in the witness protection thing, and you have quite exciting romance. I will be reading this again!
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Reviewed in the United States on September 7, 2025
★★★★★ 5
Cute and easy read
Format: Kindle
The story is cute and easy read.....just a slight bit of drama...nothing to heart wrenching. Characters are cute and very likeable...some spice....
Good ready if you just need a quick and easy story with mild spice
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Reviewed in the United States on March 5, 2025
★★★★★ 4
Thrilling Fusion of Suspense and Sapphic Longing: Blood and Muscle Delivers with Depth and Desire
Format: Kindle
Elle Alexandra delivers a masterfully woven tale of suspense and romance in Blood and Muscle, a novel that seamlessly balances pulse-pounding tension with deeply felt emotional intimacy. Set against the stark yet breathtaking backdrop of Cedar Falls, Wyoming, this story immerses readers in a world where danger and desire are inextricably linked, and where trust is both a luxury and a necessity.
At the heart of the novel are Ava Bedford, a reluctant fugitive thrust into witness protection after witnessing a murder tied to a powerful political figure, and Sheriff Lou Harman, a woman whose life is defined by structure, solitude, and an unwavering sense of duty. Their dynamic is as compelling as it is electrifying, Ava’s sharp wit and warmth slowly chipping away at Lou’s carefully maintained defenses, creating a relationship that simmers with slow-burning intensity.
Alexandra’s character work is nothing short of exquisite. Both Ava and Lou are richly developed, imbued with layers of vulnerability, resilience, and undeniable magnetism. Their interactions feel organic, their chemistry electric, and their individual arcs deeply satisfying. The town of Cedar Falls, too, becomes more than just a setting, it breathes with its own quiet menace and rugged charm, a place of both refuge and lurking danger.
The novel’s pacing is expertly handled, with the tension mounting in a way that keeps readers perpetually on edge. Alexandra deftly balances moments of tender introspection with sequences of gripping suspense, ensuring that neither the romance nor the thriller elements overshadow the other. The prose is fluid, evocative, and often poetic, drawing readers deeper into the world she has so carefully crafted.
If there is one minor drawback, it is the at-times subtle transition between the protagonists’ perspectives. While both Ava and Lou’s voices are distinct, a more clearly delineated shift in POV would have further enhanced the reading experience, offering even greater immersion into their respective inner worlds. However, this is a small quibble in an otherwise exceptional novel.
Blood and Muscle is a triumph, an intoxicating blend of romance, intrigue, and raw emotion. It is a testament to the power of resilience, the fragility of trust, and the courage it takes to love in the face of uncertainty. Elle Alexandra has crafted a story that lingers long after the final page, making this a must-read for fans of sapphic fiction who crave both heart-pounding suspense and soul-stirring romance.
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Reviewed in the United States on March 11, 2025
★★★★★ 5
🩶
Format: Kindle
🩶Small town
🩶Opposites attract
🩶Sapphic romance
🩶Witness protection
🩶Sheriff/ waitress
🩶Femme/femme
Ava witnesses a murder of a congressman and is forced into witness protection and relocated to Cedar falls. This is where she meets sheriff Lou. Chapter 1 had me quickly drawn in as it starts with Ava witnessing the murder after her shift. The tension was palpable. The writing was perfect.
I really enjoyed this book. The crime/mystery along with the romance made the story intriguing. Lou and Ava slowly start to develop a beautiful relationship with undeniable chemistry. There is just enough angst that had me stuck wanting to know how the book was going to end. Towards the middle of the book we get Lou’s backstory which pulled at my heartstrings. She went through so much as a child which explained why she was so closed off as an adult. The spice was sweet and well written. This book gave me all the feels. It’s fun, mysterious, intense, emotional and hot. I feel so much happened in 300 pages and I still craved more. The ending was wrapped up perfectly with a HEA. Lastly, I was so happy to see chapters titles! Seems to be a rare occurrence these days.
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Reviewed in the United States on April 14, 2025
★★★★★ 5
Perfect debut novel!
Format: Kindle
I had no idea this was Ms. Elle's first official debut! Story was lovely and the flow harmonious...the only indication of the author being British was the reference to an already described sweater as a jumper...but since I love British authors it only added to the charm. No real angst and not really what I would classify as a slow-burn (rather a realistic relationship timeline) made for a quick read! Can't wait to read what she writes next!!
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Reviewed in the United States on April 9, 2025