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Description
Rat RAP2B 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 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 capture antibody against the Ras-related protein Rap-2b (RAP2B). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of Ras-related protein Rap-2b (RAP2B) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Ras-related protein Rap-2b ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | RAP2B (RAP2B), also known as Ras-associated protein Rap-2b, is a member of the RAS oncogene family. It is encoded by the RAP2B gene. It belongs to the Ras-associated protein family. This intronless gene belongs to the RAS-related gene family. The proteins encoded by these genes share approximately 50% amino acid identity with classical RAS proteins and share many structural features. The most significant difference between RAP and RAS proteins is their 61st amino acid: glutamine in RAS is replaced by threonine in RAP proteins. | |||||||||||||||||||||||||||||||||
| 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 19 reviews
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Product Reviews
★★★★★ 5
Fascinating Insights into the Origins of American Racism
Format: Paperback
This is a fantastic, must read book for anyone interested in the origins of American racism. Morgan recounts the cultural, economic and political evolution of the 17th and early 18th century Virginia, and with it, makes comprehensible the reasons why racial slavery emerged as an integral component to the development of the white community's pre-revolutionary ideals of independence and liberty.
At the founding of the Jamestown colony in 1607, Virginia offered vast tracts of land available to anyone willing to make the trip and who could survive their first season (or two or three) in the New World. Unlike in England where opportunities for land ownership were constrained, the fact that Virginia land was to be had for the taking made the economic equation simple - more labor = more profits. To provide this labor, England's surplus poor (of which there was an overabundance) were sent to Virginia as indentured servants for a period of four to seven years in order to work off the costs of their relocation. Once their indenture period was over, they were free.....and poor. Over time, as established interests grabbed more and more of the land, opportunities for released bondsmen decrease, essentially creating an ever-growing class of destitute (and thoroughly despised) whites who threatened the social and political stability of the colony. Racial slavery was introduced over time to stem this proliferation of poor whites, who, after having served the term of their indenture, were free to be a "blight" on the community.
These planter elites were also constantly at political war with a succession of governors appointed by the crown to manage the affairs of the colony in a manner most beneficial to the king. By enfranchising poor whites and enlisting their support for the colonial assembly, the elites were able to exercise political power over affairs of the colony in a manner most beneficial to the colonists, rich and poor alike. The result of these forces caused a major adjustment in white social strata - the role of detested poor who would only work under the threat of the lash was imposed upon enslaved blacks, and poor whites were elevated to the level of political partners with the elites. This simultaneously endowed all whites with a fierce sense of entitlement over their political rights and the prerogatives of power on the one hand, and contempt for their black slaves on the other. Liberty and equality came to be seen as inalienable birthrights while slavery was the means by which the "shiftless, lazy, indolent" poor could be transformed from burdens on society to positive (albeit brutally coerced) contributors. In other words, Virginia whites came to think of blacks with the same sense of scorn and contempt that English aristocrats held for the poor in England while, at the same time, assuming as a birthright the same sense of political entitlement enjoyed by the elite class in England. It was this, to our modern eyes, bizarre combination of egalitarian and tyrannical ideals that informed and inspired Jefferson, Washington and Madison (among others) as they participated in the formation of what would become the United States.
The implication of this history on modern political discourse is obvious. Those who today passionately cite the liberty-loving ethos of the founding fathers while simultaneously exhibiting contempt for the poor are only looking at one side of the equation. For the Virginians, slavery and liberty went hand in hand; without the one there could not have been the other. A full, rich and nuanced understanding of our heritage compels us to recognize the human inclination to despise and exploit the powerless with the same vigor and passion that we celebrate the ennobling power of freedom.
On a final note of criticism - while the book does a masterful job of making the origins of colonial racism comprehensible, it does so at the expense of "black experience" narratives. The story addresses issues of slavery only to the extent of discussing laws passed throughout the pre-revolutionary period in order to institutionalize it and the effect these laws had on the attitudes of whites towards blacks. I started the book expecting a far deeper dive in this area, and was disappointed by how little was presented concerning the evolution of slavery throughout the 17th century from a black perspective. After having read the book, I concede that this deeper dive was not strictly necessary in order for the author to prove his thesis, yet it would have been a stronger work had greater efforts in this area been made.
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Reviewed in the United States on February 23, 2013
★★★★★ 5
Great Historical Reading
Format: Paperback
I found "American Slavery, American Freedom" to be a thought-provoking book that contained a great deal of useful information. I wrote in the margins of the book, took notes, and highlighted entire pages.
"American Slavery, American Freedom" was well-written and enjoyable to read.
I had read countless books on slavery over the years. This book did not focus primarily on slavery. A detailed description of the steps and events that led to the creation of the Commonwealth of Virginia can be found in "American Slavery, American Freedom."
The history of Virginia is characterized by slavery and servitude. Since many of the books I had read on slavery lacked a compelling backstory, I found this book refreshing.
As far as I can tell, the author denied or downplayed the fact that Thomas Jefferson fathered many children with a slave named Sally Hemmings. The author probably worked on this book for years before its publication in 1975. There was a possibility that Edmund Morgan did not want to write about any "touchy" topics.
"American Slavery, American Freedom" was a pleasure to read. I would recommend it to others.
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Reviewed in the United States on September 30, 2020
★★★★★ 4
Disturbing Questions
"Racism became an essential, if unacknowledged, ingredient of the republican ideology that enabled Virginians to lead the nation." writes Edmund S. Morgan in 1975, and ends this book with the rhetorical question: "Is America still colonial Virginia writ large?"
These are deeply disturbing questions - questions one is compelled to ponder as one reads this lucid and dispassionate presentation of the how primitive accumulation in Virginia at the beginning of the 17th century was replaced a century later by an orderly and opulent society based on slavery. The answer to such questions is not made easy by the realisation that the only other successful republican experiment - the Athenian democracy - blossomed too on a bed of slavery.
Do these questions matter today? Have we not moved on from racism? I'm afraid not. Again the voice of Morgan: "In the republican way of thinking, zeal for liberty and equality could go hand in hand with contempt for the poor and plans for enslaving them." Sounds eerily familiar? Just as today's language used to describe terrorist threats is redolent of the rhetoric that once surrounded the lynching of black bodies. Racism (albeit globalised) is re-visiting the land today, and so are republican virtues and values.
The book is long, and in some ways, too detailed. Morgan delights in the telling particular, and at times one wishes he would not linger on some specifics. But this has a purpose. He wants to show the imperceptible and surreptitious mechanisms by which a society acquires its ugly and immoral traits until they become so natural as to be invisible. Step by step, event by event, law by law a construction emerges that would have horrified its founders. Yet, at the time, it seamed the logical, and the right thing to do.
A strong point in Morgan's narrative is the links he highlights between the developments in Virginia and the Britain's commercial interests, migration policies, population growth and control, state revenue, and political history or thought. One can better appreciate the import of Virginia for Britain and the mother country's fixation and fascination for the North American colonies.
Brash and brutal, Virginian slavery stood openly as godmother at the foundation of the American Republic. Other aspects of slavery also contributed significantly - but as they were indirect, they remained veiled and are hardly recognised even today. New England benefited greatly from its cod trade to the Caribbean, where the product that was found to be unfit for European markets was fed to the slaves, thus freeing up land that otherwise would have been used to sustain them. When will we get a total picture of slavery's import for America's economic foundations?
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Reviewed in the United States on July 8, 2003
★★★★★ 5
how a country could develop a "national character" founded on the love of liberty while simultaneously importing thousands and t
Format: Paperback
This book lays out hte paradox, how a country could develop a "national character" founded on the love of liberty while simultaneously importing thousands and thousands of bondsmen to provided the "free people" with the necessities of life: i.e., why slavery was necessary to support the kind of freedom the white folk wanted to become accustomed to.... and implicitly, why the industrial revolution finally changed the hearts and minds of enough Americans to make slavery seem unnecessary and therefore, if was no longer a necessary evil, why it had to be overthrown.
Morgan writes objectively -- but his feelings are always detectable through his writing style, which is perhaps the best academic English to be found anywhere. I found it gripping. The book was published in 1972, and has doubtless been corrected by many subsequent researchers in some of its particulars -- but it was the fountainhead for a new way of understanding American history that young people all have learned about in high school, but which many baby-boomers have never seriously encountered.
Reading it accomplished a MAJOR retrofit in my sense of how the USA got to be the way it is today. Not to put too fine a point on it, the Tea Party and many trump supporters seem to adhere to the values of the original American Republicans [and to think that Black folk should be pushed back to a place where their feelings don't matter], and to long for a return to the status quo ante -- with ante referring to a time long LONG ago
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Reviewed in the United States on August 15, 2016
★★★★★ 5
U.S. American Genesis
Format: Kindle
Kindle edition worked well. Very interesting and insightful read by a first rate historian. Tells the story of how our ancestors transitioned from Englishmen to Americans. A book well worth taking the time to read.
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Reviewed in the United States on June 8, 2022