Archive for August, 2009

Allele Pre-packaged, Titer-determined, Validated iPS Generating Lentivirus Particles

Induced pluripotent stem cell (iPSC) has been an important research area in the past 3 years. These cells have also provided unprecedented possibilities to study cell differentiation and tissue development to biologists in many fields. Allele pre-packaged iPS lentiviral particles are validated, ready-to-use, high quality reagents for any laboratory to create iPS cells. A straight-forward and optimized protocol is also provided for immediate use of these products. iPC cells can be produced typically in 2-3 weeks; but when used in combination with shRNA against p53 (also available from Allele), iPSCs can be produced in just about 5 days!

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Immunoprecipitation Tags

Immunoprecipitation is a process of isolating a protein as an antigen by using antibodies against it. It is a powerful tool for studying proteins in biological samples and, in case of Co-IP (meaning immunoprecipitation of complexes containing a known antigen), for analyzing protein-protein interactions. Similar technologies such as chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP), or crosslinked and iImmunoprecipitation of RNA-protein complexes (CLIP) aid analysis of protein-DNA or protein-RNA interactions.

The major obstacle for achieving effective immunoprecipitation is the difficulty of finding usable antibodies against a target of interest. A common practice is to use tags that are fused to the C- or N-terminus of the target protein, thereby any validated, commercially available antibody can be used for co-IP in different experimental systems. However, caution must be exercised against potential interference of biological functions from the added tags. In general, one should choose tags that have been tested in many situations and proven non-interfering; still, each biological system is different. Independent validation or supporting data should be used when interpreting results from tag-based co-IP.

Tags are often selected based on high quality and commercially available antibodies. Most commonly used tags include: FLAG, Myc, HA, V5, T7, and His, which are quite small in size and in theory less likely to interfere. GST and GFP are in between 20-30kDa, but they are well documented to form self-contained and stable structures independent of their fusion partners and proved to not interfere in many cases. GST can bind to glutathione beads directly, therefore a top choice for pulldown experiments. GFP or other FPs as tags have the advantages of being also a visualization module to follow the protein both inside cells and during pulldown. However, previously available anti-GFP antibodies, either polyclonal or monoclonal, are not comparable to those against other tags, thereby limiting the use of GFP as fusion tag in pulldown experiments.

GFP-Trap, a recent addition to anti-tag antibodies, is an E. coli expressed, single domain fragment derived from camelid heavy chain antibodies (VHH antibodies) with much higher stability, specificity, and affinity, making GFP based pulldown quantitative. This recent advancement should make GFP in line to become the most suitable tags for many aforementioned precipitation experiments.

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Francis Collins On the Job

Dr. Collins did a town hall meeting style announcement his first day as NIH Director on Aug 17th, 2009. He laid out his view for the NIH: more funding (good), encouraging young scientists (good, average age for first own funding for US biologist is 42, not good), and staying open in communication with society it is serving.

The NIH has $30.9 billion budget for 09 and 2010 thanks to the stimulus addition of $10 billion/year. However, it will feel dried up after two years if the budget plan remains as is. The Obama administration does not seem to want increase the basic research but instead focus more on health care management.

Collins is a well admired director and established scientist. However, it may be a little concerning that he might be too much into “big science” and organized efforts. I don’t know what they teach in graduate classes now but from what I was told 20 years ago curiosity-driven science is the best science and that was what got the US to the dominant leadership in biomedical fields.

Talking about nurturing young scientists, big programs and big labs controlling most grants by proposing big science seem trendy these days. The fight to become one of the big guys in a small, crowded field is a really daunting path for young researchers to tread. The big guys have the say from publication to funding and often times the unpleasant thought and bitter taste of competing against a scientific juggernaut turn young researchers away.

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Economy and Your Research: Carpets and Oligos

Do you believe in six-degrees of separation? If you really don’t care how close you are related to Roger Tsien or Bill Gates or the dean of your graduate school, maybe you are still curious about how the economy downturn, oil production, and floor carpet production got to do with you–not just in the sense how the job market is shaping up, but also how your lab research budget and how your DNA oligos are served.

To illustrate how events far and away can influence your daily activities, just use oligos as an example. Starting in 2008 when the oil price was still near its peak (remember paying $4+/gallon?), it became too expensive for carpet producers to continue using petroleum for manufacturing carpets. They switched to some other source or halted business altogether. Side effect was production and supply of Acetonitrile (ACN) dried up. Yes, one of the most commonly used organic solvent is a by-product from making carpets. That, combined with facility shutdowns in Northern China in preparation of the summer Olympics (for clean air) and in Florida by a major hurricane, the price of 4 liters of Acetonitrile changed from ~$40 to about $400 plus lots of begging. This event alone pushed individual customer based (as compared to large scale or prefabricated) oligo businesses like Allele’s to be at a loss.

Eventually the situation changed, price went back to about $90/4L, but not before a long period when Acetonitrile was completely unavailable and alternative solvent had to be used. Long story short, that was some storm to whether! If you didn’t feel it in price or service from Allele Oligo, good, that means we did a fair job shielding the wind and shouldering the pressure from the collapsing roof.

Everything really is connected, sometimes by a few degrees less than you would imagine.

Allele’s mottos: care about the environment, help everybody whenever we can, do the right thing even when nobody is looking, have fun, and contribute to the good of mankind through science and innovation.

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Friday, August 14th, 2009 oligos and cloning, State of Research No Comments

Camelid Antibodies

When it was discovered that animals in the camel family produce antibodies with no light chains, the idea that a single-domain fragment can bind as well as a full 4-chain antibody formed a breakthrough. So far it has been a relatively less known one.

Smaller antibody fragments have been tested for therapeutic uses because classical IgG antibodies are too bulky to penetrate tissues well, and very expensive to produce. Different combinations of antigen-binding variable regions are used, e.g. scFv, Fab, diabody, all to some degree of success. In comparison, the N-terminal domain of camelid antibodies, termed VHH domain (nanobody, VHH antibody), represents a naturally evolved, only 13-15 kD in size, fully functional target binding fragment with many advantages.

The only other known species outside camelidae family that has heavy chain antibodies is particular cartilaginous fish, nurse shark. Although the arrangement of CDRs is somewhat different between the camel and shark heavy chain variable regions, they share many characteristics such as extremely high stability (maintaining functions after100 C heat and extreme pH treatment).

Accumulating reports have demonstrated the therapeutic potentials of camelid antibody-based fragments in treating cancer, neural diseases, even use in hair dandruff preventing shampoo. For basic research, the tiny antigen binders can be used as tools for quantitative pull down with unmatched efficiency, recognizing previously inaccessible enzyme cleft as antigens, and providing libraries for binding partner selection.

Allele Biotech has been working on display antibody selection from its early days through an NIH grant, and recently carried out an NIH/NCI contract for scFv yeast display.

Check out Allele’s current Camelid antibody products:

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