This is a simple method for rapid preparation of recombinant adeno-associated virus (rAAV) stocks, which can be used for in vivo gene delivery. The purity of these vectors is considerably lower than that obtained by either CsCl gradient centrifugation or by combination of iodixanol gradient ultracentrifugation followed by column chromatography.
This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.
Differential detergent fractionation of cells is a rapid method for extraction of cytoplasmic and nuclear proteins in preparation of an immunoprecipitation. This method can be applied for use of adherent or suspension cells and can significantly reduce nonspecific background in an immunoprecipitation by separation of cellular compartments into individual fractions. The lysis of cells by differential detergents permits the rapid extraction of proteins from the cytoplasm (digitonin), the cytoplasmic membranes, and organelles (Triton X-100), and nucleoplasm (Tween/DOC), facilitated through the use of distinct extraction buffers. Cytoplasmic and nuclear matrix proteins as well as DNA are left behind during the detergent-based extraction.
The only way to solubilize many antigens for immunoprecipitation is by denaturation. This cell lysis protocol is ideally suited for this purpose to release proteins from complex structures or reveal antibody epitopes hidden within native proteins. Short linear epitopes may not be accessible to antibodies within the native tertiary and quaternary protein structures, but they become exposed upon the unraveling of proteins, exposing their secondary structure. Antibodies otherwise not suitable for the immunoprecipitation of proteins prepared under nondenaturing conditions are now able to bind these antigens of interest in cell lysates prepared under denaturing conditions. These antibodies may also work well for immunoblotting purposes when the protein target is completely denatured. Harvested cells in this protocol are washed in tris-buffered saline (TBS) before lysis in 2% sodium dodecyl sulfate (SDS)-containing Lysis buffer for 10 min at 100°C. The resulting sample is diluted 20-fold in TBS to reduce the SDS concentration to ≤0.1% before the addition of an antibody for immunoprecipitation. Addition of 2% bovine serum albumin (BSA) or 0.1% Nonidet P-40 to the TBS before an immunoprecipitation, respectively, ensures either removal of SDS from the target protein or retaining denatured proteins in solution.
In the early 20th century, Native people responded to the proclamation of Mother’s Day with powwows, ceremonies, rodeos, feasts, and songs
The Kent State shootings further widened the chasm among a citizenry divided over the Vietnam War
The institution took decades to come to grips with the trauma of the killing of four students 50 years ago
As Babe Ruth was emerging as baseball's great slugger in 1918, he fell sick with the flu
Sheltering in place doesn’t mean you can’t study up for your next European adventure
Plague, war and politics have altered the yearly pilgrimage to Mecca throughout history
Governments around the world grapple with how to deliver important guidelines on minimizing the spread of COVID-19
During his 20-year professional career, his boisterous style endeared him to fans but rankled traditionalists
Seventy-five years after a memorable mission, the B26 bomber 'Flak-Bait' undergoes preservation at the National Air and Space Museum
From kitsch to cool, ride the waves of undulating popularity of a tropical fashion statement
In a nation under quarantine, chronicling a crisis demands careful strategy
Follow the Frenchman who remade the woods surrounding a royal estate into the world’s first nature preserve
In the new miniseries, feminist history, dramatic storytelling and an all-star-cast bring the Equal Rights Amendment back into the spotlight
The Beatles guitarist visited his sister in southern Illinois just months before he'd become world famous
A new book charts the history of counting the public, from the ancient censuses in Rome to the American version of decennial data collection
Tensions leading to the split, announced 50 years ago today, had been bubbling under the band’s cheery surface for years
The third installment in our weekly series spotlights titles that may have been lost in the news amid the COVID-19 crisis
With the release of Trolls World Tour, and a new generation entranced by the ugly-but-cute toy, it appears the troll's lucky streak lives on
Sketches from the workshop of French engineer Gustave Eiffel suggest a different plan for Lady Liberty’s upraised arm
Using the lunar module as a lifeboat and employing techniques never before considered, the astronauts' ordeal ended triumphantly
A new book explores how George Washington shaped the group of advisors as an institution to meet his own needs
The second installment in our weekly series spotlights titles that may have been lost in the news amid the COVID-19 pandemic
While COVID-19 has us homebound, it’s a good time to reflect on the peculiar histories of housewares we take for granted
The question of bulletproofing vexed physicians and public figures for years, before pioneering inventors experimented with silk
The first major exhibition of its kind, "Hearts of Our People," boasts 82 pieces from 115 Native women across North America
The National Trust for Historic Preservation is looking for 1,000 places tied to women's history, and to share the stories of the figures behind them
We're highlighting newly released titles may have been lost in the news as the nation endures the coronavirus pandemic
A museum director looks to the past to explain why 'Aloha' is as necessary as ever
Smithsonian curator Eleanor Jones Harvey explains why this revolutionary 19th-century thought leader is due for a reconsideration
A century ago, Susan Pringle Frost tirelessly campaigned to save these South Carolina buildings from destruction
From virtual museum tours to space exploration, ancient worlds and natural phenomena, this hub has you covered
In the hours after disaster struck Anchorage, an unexpected figure named Genie Chance came to the rescue
It all began when three frustrated women sought the no-bounce zone
Tour a gallery of presidential portraits, print a 3-D model of a fossil or volunteer to transcribe historical documents
Historian Ellen Carol DuBois chronicles the twists and turns of the 75-year-path to securing the vote for women in her new book
In 1919, a second wave of cases of the previous year's flu lead to the sudden death of the hockey championship
The hair-care magnate at the center of the new Netflix series 'Self Made' left her imprint on the city where she launched her career
Philip Roth's classic novel, newly adapted by HBO, envisions a world in which Charles Lindbergh wins the 1940 presidential election
History remembers the English queen as a murderous monster, but the real story of Mary I is far more nuanced
A sled in the Smithsonian collections marks the historic race
A Brown Bear takes a quick dip while hunting for salmon at Anan Creek Bear Observatory in SE Alaska.
Savage’s Cochran Frog (Centrolene savagei) looking from the edge of a leaf at cloud forest, 1640 masl, this species is endemic to Colombia.