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Description
Human UBA2 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 ubiquitin-like 1-activating enzyme E1B (UBA2). 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 ubiquitin-like 1-activating enzyme E1B (UBA2) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Ubiquitin-like 1-activating enzyme E1B ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Ubiquitin-like modifier activating enzyme 2 (UBA2), also known as ubiquitin-like 1 activating enzyme E1B (UBLE1B) or SUMO-activating enzyme subunit 2 (SAE2), is an enzyme encoded by the UBA2 gene. It regulates protein structure and cellular localization through the post-translational modification of proteins by incorporating the small protein SUMO, or sumoylation. SAE1 and UBA2 form a heterodimer that functions as a SUMO-activating enzyme for protein sumoylation. Disorders associated with it include chromosome 19q13.11 deletion syndrome. | |||||||||||||||||||||||||||||||||
| 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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4.6 ★★★★★
Based on 13 reviews
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Product Reviews
★★★★★ 5
Essential History for Retaining Reason and Rationality for Guiding the Nation
Awesome book on the History of people, themes and big money, corporate used in an attempt to make one religion the National Religion while neutering the First Amendment. It is true to it's title in detailing how Corporations and Religion came together to fight the New Deal of FDR. These two self serving entities fought for control of the people but did almost nothing to avert wide spread hunger and homelessness in the depression. The fortunes of corporations and insistence of religions on controlling the minds of people to produce jobs in their own occupations outweighed the immediate needs of the jobless and homeless. The opulent lifestyles of the big religions and their organizers came first over the health and welfare of the country, just like it had in Medieval Europe, where the Aristocrats and the Christian Churches ran the Continent. These two institutions were responsible for keeping Democracy out of Europe and ultimately creating the Soviet Revolutions in Russia that attempted to spread to the rest of the World. What is clear, from many books on this subject, that "Under God" means being subservient to God, the word of which comes from God's messengers. Subservience Under God means that when the opinions of the People differ from those of God as told through the Messenger, then God wins and the People Shut Up. Those that don't face stiff penalties. Fabulous History that everyone should know if they want to perpetuate a Democracy. The Roman Empire was "One Nation Under God", because the Christian Religion was the only legal Religion and it had only One God - no others were allowed and this was enforced with the sword. Rome will return to America if we are not careful.
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Reviewed in the United States on October 22, 2015
★★★★★ 3
Stranded on third base
Format: Hardcover
Prof. Kruse wanders away from the promise of the subtitle of his book--"How corporate America Invented Christian America." He almost gets there when he describes the ideological opposition of the wealthy to F.D.R's "New Deal." and their caricature of the "social gospel" as Communist propaganda. His description of Truman's encounter with Billy Graham is wonderful. For a book published in 2015, Kruse makes only slight mention of the fact that the politics of money and religion in contemporary times becomes more pernicious every day. The main body of the book drifts into redundant detail about "who prayed with who" and the spiritual influence of prominent evangelists on presidents from Eisenhower to Bush with only faint reference to their ties with the Capitalist agenda. He portrays wealthy Christians as true religious zealots when, in fact, they and many of the politicians they supported were likely hypocrites using religion as a wedge issue to increase their power and pursue their agenda of unregulated Capitalism. It is remarkable that Kruse entirely fails to mention the Koch brothers--billionaires who bankroll countless ultra right-wing politicians with evangelical agendas, while they, themselves, are apparently Ayn-Rand-style libertarian atheists interested only in a social-Darwinian, Capitalist heaven. Prof. Kruse leaves us stranded on third base, when he could have hit a home run!
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Reviewed in the United States on May 7, 2015
★★★★★ 5
Horrific Voter Suppression
Format: Paperback
This is a very well documented book about voter suppression in America. It was interesting to see that before the Jim Crow era, black people voted, were elected to office, got educations and did fairly well. With Jim Crow, horrific voter suppression began (lynchings, etc.). In the early days it was the Southern Democrats and the KKK. As time went on the methods simply became more devious. In the 1930s FDR began attempting to attract black voters to the party. By the end of WWII we had "Dixiecrats". By the time the time the Civil Rights Act was passed, the transition was complete and those Southern Democrats were now members of the GOP. (Much of that bit of history is not in the book, but you'll want to look it up.) The GOP is now the master of voter suppression, and you can see it pretty much everywhere but especially in Georgia, Texas, Minnesota and many southern states. The book is chock full or examples, case histories and stories of resistance. It made me want to sell my house and give the money to the ACLU, NAACP, and SPLC to defend our most precious right (not privilege) to vote.
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Reviewed in the United States on October 4, 2020
★★★★★ 5
This is a MUST read!!
Format: Kindle
This book is a must read! I was just not aware of the extent of the planned, organized, deliberate and insidious actions that the Republican Party has taken to suppress votes from the disenfranchised and the impact that has on our Democracy. I am stricken by the horror of what has been happening now for YEARS and is still going on today!! Carol Anderson has compiled hard data to demonstrate the pervasiveness of the many voter suppression steps that have been taken across many states, primarily in the deep south, but not exclusive to the south. It’s very difficult to take reams of data and put it into a coherent, logical sequence that can be easily understood but she is masterful at making her book eminently readable. This is a very data driven book but I had no trouble getting through it. The conclusion did have some hopeful information relaying steps that some states are taking to increase voter participation which did give me some hope. I am so glad that I read this and I think we all should be informed on this issue as it is central to our state as a country.
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Reviewed in the United States on April 30, 2021
★★★★★ 5
Great book but disappointing kindle experience
Format: Kindle
A must-read for anyone who wants to understand the troubled and racist history of voting rights in the US.
My only complaint is that the Kindle version of this book messes up the footnotes. The great thing about reading on the Kindle is that you don't have to flip back and forth to check the footnote cites; you only have to click on them. Sadly, the footnotes aren't correct in the Kindle version of the book. They somehow were shifted by one, as I was able to confirm in my hardcover copy, completely ruining the experience if you wish to read more about a specific incident. This is inexcusable in my mind, but I won't hurt the rating for this book by taking off a star.
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Reviewed in the United States on January 5, 2021