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The Updated Technique in De Novo Antibody Sequencing

Antibody Sequencing has been a crucial technique in researching and developing various antibodies. There are many manufacturers who are dedicated to make this technique much better.

To differ themselves from the crowd, they have develop something unique, Creative Biolabs, for instance. Their antibody sequencing method is quite unusual.

De Novo  Sequencing of the CDR3 region

While the CDR3 of the light chain is mostly encoded by the germline sequences, the CDR3 of the heavy chain is usually not available in databases. It is encoded by the so called D-segments but these are modified by nucleases and terminal transferases. Typically, only 1-4 AA of a D-segment remain in the matured antibody. The rest of the D-segment is “artificial” and has to be sequenced de novo.

Creative Biolabs generates many overlapping peptides during the fragmentation process, enabling us to sequence very long stretches of unknown amino acids. The high quality of MS/MS spectra in combination with intelligent data mining, allows them to read the CDR3 like a book. The technique is so powerful that they were able to sequence a 20 kDa protein, which had no homologue in the database.

Isobaric amino acids

In contrast to other MS based methods, they can discriminate most isobaric amino acid combinations. E.g.:
• W can be distinguished from GE, AD and SV (by mass difference)
• R can be distinguished from GV (by mass difference) 
• Q can be distinguished from GA and K (by fragment spectra and mass difference) 
• N can be distinguished from GG (by derivatization and fragment spectra) 
• Leucine and isoleucine cannot be distinguished. However, most of these positions can be determined using the corresponding germline sequence (see figure 1). As antigen binding is mostly mediated by salt bridges and hydrogen bonds the impact of Leu/Ile is usually negligible. 

Sequencing of fluorochrome mABs, IgMs and other non-standard antibodies

 Their method is usually not affected by small ligands (FITC, Biotin, Alexa) coupled to antibodies. Larger protein ligands make sequencing slightly more difficult. However, this can be compensated by using a higher protein concentration, because sequencing quality is dependent on sample amount.

 IgMs can usually be sequenced like normal IgGs. Slightly more sample may be required. As the constant region is modified by several glycans, which cannot be cleaved by PNGase F, they can only guarantee complete coverage for the variable part of the antibody. Their method works best with mouse and rat antibodies. However, ~50 antibodies from rabbit hamster and lama have been sequenced successfully. Besides, many older hybridoma produce 2 light chains (one is a nonsense light chain). These mixtures can usually be sequenced, even when the nonsense chain is in twofold molar excess.

Learn more about unique de novo antibody sequencing.

What You Need to Know About scFv/Fab

Fragment antigen-binding (Fab fragment) is a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. Single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids.

Fab fragments differ from scFv's as they contain both variable domains and constant regions, thus are more complex in construction and ultimate expression. Creative Biolabs is an expert with many years of experiences in antibody production. It has established a solid platform for scFv and Fab constuction. The same heavy and light variable chains used for scFv construction can also be used in the construction of Fab.

There are three routes to have a scFv or Fab target a specific antigen in Creative Biolabs. The first approach is to generate a mouse hybridoma clone, then convert full IgG [or IgM] into a scFv or Fab; the second one is to create an immunized phage display scFv or Fab mouse library, then use the antigen to screen the library; the third method is to use the antigen to screen a premade scFv or Fab antibody phage display library [usually a human one] to get scFv or Fab clones directly. We are also able to convert chicken IgY into scFv or Fab fragments.

Learn more by visiting scFv/Fab Construction.

Know When Milky Way Collision Occur

NASA scientist have found proof that our Milky Way had an encounter with a small galaxy or massive dark matter structure perhaps as recently as 100 million years ago. Are you also interested to know how galaxies form, evolve and interact?
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Know more about Saturn’s 2010 Great White Spot storm that has set a new record for largest temperature change that has ever recorded for a storm on Saturn. NASA’s Cassini spacecraft, researchers spotted a massive belch of energy that sent temperatures soaring to150 degrees Fahrenheit above normal in Saturn’s stratosphere.
Robotic missions to Mars have revealed clues about the planet’s atmosphere and surface composition.NASA is investing to find and use natural resources beyond Earth. Know more about Robotic Mars Mission at Astrobiology Magazine.

Know How Origin of Earth's survived when Oxygen has Abundant

We all are aware of the fact that billions of years ago, there was very little oxygen on Earth to breathe. Scientist of University of California at Riverside (UCR) have researched when in Earth’s history oxygen may have abundant. Curious to know how origins of Earth survived?

Know More About Plenty of Water in Asteroid Fragments

Do you know new study could provide visions about the abundance of water in fragments from a famous asteroid? This research mainly focused on a mineral called apatite Want to know more about this asteroid?
The European Space Agency (ESA) has released an incredible new video showcasing images from the Mars Express mission. Want to know more about Mars mission?

Identify More about First Light At MARS

Are you aware of the MARS mission? Do you know how long does Mars mission take to start & sending back data once it’s arrived at the red planet? Read more about Mars atmosphere, wavelength, MAVEN etc. only at Astrobiology Magazines.
The Milky way steals the show from Sahara sands that make the Earth glow orange. Reid Wiseman a NASA astronaut has captured this pic from International Space Station.

Know How Cassini Looking Mysterious Feature Evolve in Titan Sea

Do you know NASA’s Cassini spacecraft is monitoring the evolution of a mysterious feature in a huge hydrocarbon sea on Saturn’s moon Titan? Curious to read more about mysterious feature?
Know more about what is mars mass balance challenge only at Astrobiology Magazine.
Interested to know where life begins on earth? Nicholas Hud and a team from the CCE(Center for Chemical Evolution) are working to understand the origins of life on Earth by studying how chain-like chemicals called polymers first came together to form RNA and DNA.
At Mars scientist are researching about MAVEN interaction of neutral gases and ions in the Martian atmosphere with the solar wind that helps to know how Mars has lost its atmosphere over time.

Discovery of a Massive Planet that makes its own Star

Explore the facts, myths, images and latest news of how a massive planet effect the behavior of its parent star. Curious to know more about this planet?
Do you know how origin of life has started in space? No, then we atastrobio.net help you to know how studying a comet can help us to learn about life in the solar system.
International Space Station photographed this nighttime image showing city lights from areas in the Gulf Coast states of Texas, Louisiana, Mississippi and Alabama.
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Fluorescent Cat. At least you will not stand on it at night ;)
Reposted fromMafea Mafea viascience science
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