October 2010

BenchTopics™ Newsletter for Protein Technologies and Advanced Cloning


Transfection

 

Don’t miss the online in vivo RNAi delivery seminar

RNAi is a powerful tool that can be used in the drug discovery process to identify new targets for drug design or to validate the target for which a drug was designed. Unfortunately, delivering RNAi duplexes to the appropriate tissue remains a major bottleneck for the development of in vivo RNAi. In this seminar, you will learn techniques and considerations for successful in vivo RNAi experiments and will get to hear how our new a new, commercially available, in vivo delivery reagent, Invivofectamine™ 2.0, helps overcome these challenges.

A Life Technologies scientist will host the online seminar, so you can attend from the comfort of your own desk.

Register to attend

Protein expression & purification

 

AcTEV™ Protease for efficient cleavage and removal of epitope tags and fusion partners

Use AcTEV™ Protease, an enhanced version of tobacco etch virus (TEV) protease, to efficiently cleave and remove solubility, secretion, detection, and purification tags from recombinant proteins. AcTEV™ Protease has the same sequence and cleavage site-specificity as TEV (Glu-Asn-Leu-Tyr-Phe-Gln v Gly). AcTEV™ Protease retains 100% activity, even after incubation for 1 week at 4°C. Under the same incubation conditions, non-enhanced TEV protease looses 25% of its activity within 24 hours (see figure).

To find all Invitrogen™ protein expression vectors containing a TEV cleavage site, browse to the Vector Selector Tool, and under “N-term Protease Cleavage” select TEV. For free, online primer design with a one-step addition of a TEV cleavage sequence to your gene, go to OligoPerfect™ Designer.

  • Effectively remove N- or C-terminal solubility, secretion, detection, and purification tags from recombinant proteins

  • Protect your protein of interest from nonspecific proteolysis—AcTEV™ Protease is manufactured to exhibit highly specific cleavage activity, without contaminating proteolytic activity

  • Easily remove AcTEV™ Protease from your digestion mix using the six-histidine tag

  • See recent publications mentioning AcTEV™ Protease (indexed by HighWire Press).

  • If you typically see low yields, investigate our pcDNA™3.3-TOPO® TA Mammalian Expression Vector for the ultimate in high-level mammalian protein expression

Electrophoresis & western blotting

 

The NativePAGE™ Novex® Bis-Tris Gel system provides a sensitive and high-resolution method for the assessment of the purity of native proteins, the analysis of native membrane protein complexes, native soluble proteins, and molecular mass estimations. The NativePAGE™ Novex® Bis-Tris Gel system is a native (nondenaturing) precast polyacrylamide mini gel electrophoresis system that operates at a near-neutral pH of 7.5. This neutral environment protects both the proteins and the gel matrix during electrophoresis, providing for higher band resolution than the traditional Laemmli system. (Figure reproduced from Kubota S et al. (2006) Proc Natl Acad Sci USA 103:8360–8365.)

NativePAGE™ Gel System
The NativePAGE™ Gel system is based on the blue native polyacrylamide gel electrophoresis (BN PAGE) technique developed by Schägger and von Jagow (Schägger & von Jagow, 1991) that uses Coomassie® G-250 as a charge-shift molecule. In standard SDS-PAGE, the charge-shift molecule is SDS. The SDS binds to the protein and confers a net negative charge, which ensures that the protein migrates in one direction towards the anode. For this technique, SDS is present in the sample buffer and running buffer. Similarly, in BN PAGE, the Coomassie® G-250 binds to proteins and confers a net negative charge while maintaining the proteins in their native state. The G-250 is present in the cathode buffer to provide a continuous flow of G-250 into the gel, and is added to samples containing non-ionic detergent prior to loading onto the gel. The gels themselves do not contain any G-250.

The binding of G-250 to proteins offers the following advantages resulting in high-resolution native electrophoresis (Schägger, 2001)

  • Proteins with basic isoelectric points (pI) (that would normally carry a net positive charge at neutral pH) acquire a negative charge, forcing all proteins in the mixture to migrate towards the anode.

  • Membrane proteins and proteins with significant surface-exposed hydrophobic area are less prone to aggregation as G-250 binds non-specifically to hydrophobic sites conferring a negative charge to those sites.


The NativePAGE™ Novex® Bis-Tris Gel system is suited for:

  • Analyzing native membrane protein complexes or soluble protein complexes

  • Determining the purity of native proteins, and estimating molecular masses of native proteins and complexes

  • Performing two-dimensional native/SDS-PAGE to resolve complex samples

  • Analyzing protein complexes purified using the Invitrogen™ NativePure™ Native Complex Purification System

  • Performing in-gel or solution activity assays

Read our NativePAGE™ application note.

View journal articles referencing NativePAGE™ Novex® Pre-Cast Gels (indexed using HighWire Press).

To see additional details and to order see

Protein interaction

 

ProtoArray® human protein microarrays continue to help in discovery of new disease markers

Auto-antibodies are ideal molecules for establishing new diagnostic and efficacy biomarkers. The ProtoArray® human microarray offers a printed surface containing over 9,000 full-length human antigens, representing a wide spectrum of potential auto-antigens from a variety of protein classes. The ProtoArray® microarray’s success in identifying auto-antibody–based biomarkers was recently demonstrated in a publication by Cell Gensys Inc. The researchers examined changes in patient immune profiles on the array before and after immunotherapy in prostate cancer patients. The researchers identified several auto-antibodies that were elevated in response to the treatment that may serve as viable biomarkers for positive therapy response in larger patient samples.

To find out more about ProtoArray® microarrays and biomarker identification, visit us online at www.invitrogen.com/absoluteid. We offer catalog ProtoArray® microarrays for purchase, and we can also create custom microarrays in our full-service laboratory to meet your experimental needs.

Advanced cloning

Life Technologies™ is proud to announce the launch of a new set of cloning tools that will revolutionize the way you perform research. With the addition of these products to our market-leading cloning tools portfolio, you’ll be able to access novel and innovative cloning platforms and mutagenesis solutions in your laboratory. These new cloning tools offer significant advantages in everything from constructing simple DNA vectors to assembling complex pathways or mini-chromosomes:

Easy and Powerful — Clone up to 10 DNA fragments, with the sequence of your choice, simultaneously in a single vector (up to 110 Kbp); no restriction digestion, ligation or recombination sites required

Flexibility — Use our linear vectors or a vector of your choice, clone pre-existing DNA fragments that have no end-homology without further modifications

Precision and Efficiency —Designed to let you clone what you want, where you want, in the orientation you want, and achieve up to 90% correct clones with no extra sequences left behind

Also available is the new DNA Oligo Designer, an intuitive, web-based tool to guide your design. The tool minimizes the planning time required for designing the PCR primers, creates the end-terminal homology required for seamless assembly,

identifies potential pitfalls linked to your specific sequences, and performs in silico cloning using your sequences. The DNA Oligo Designer also provides you with a graphic representation of the final assembled molecule as well as a downloadable GenBank file compatible with VectorNTI® software.

Learn more about these new tools today!

siRNA

 

Ambion® In Vivo siRNA

Ambion® In Vivo siRNA molecules are chemi¬cally modified, 21-mer double-stranded siRNAs that are recognized by the RNA-induced silencing complex (RISC) to mediate inhibition of a target gene. Proprietary chemical modifications allow Ambion® In Vivo siRNAs to overcome many in vivo–specific obstacles, ensuring their effectiveness and stability. Ambion® In Vivo siRNAs are at least one hundred times more stable in 90% mouse serum than unmodified siRNAs.

As the field of RNA interference (RNAi) matures, researchers are becoming increasingly interested in discovering the in vivo effects of gene silencing. However, due to the presence of high levels of nucleases in blood and other biological fluids, in vivo RNAi experiments are more challenging than those performed in vitro. This increased challenge necessitates siRNAs that are specifically developed for in vivo applications, incorporating optimal sequence design features, appropriate chemical modifications, and the use of high-quality materials. We are now able to offer the most advanced siRNAs for in vivo applications—Ambion® In Vivo siRNAs.

Ambion® in vivo siRNAs, the new standard for in vivo RNAi applications, offer:

  • High stability against nucleases—siRNAs reach their target intact and ready to initiate knockdown

  • No induction of the interferon response—correlate phenotypes with knockdown activity instead of toxicity

  • Easy tracking of administered siRNAs—measure biodistribution effectively using TaqMan® Assays

  • Combined knockdown effectiveness and stability—no need to compromise one for the other

Interview

 

Each month we feature a different Life Technologies employee and share his or her story. This month, we interviewed William Staerker, Global Market Development Manager, Molecular Biology Reagents.

  1. Name—Current position
    William Staerker, Global Market Development Manager, Molecular Biology Reagents

  2. Formal education
    B.S. Biochemistry, Berry College
    M.S. Biochemistry, The Ohio State University
    M.B.A. Marketing, Cornell University

  3. Work experience—focus on leap from academic to business
    After finishing my MS I worked as a staff scientist for Nestle Purina in the department of molecular nutrition. Nestle is a marketing-centric organization, and I was impressed by the role marketing had in everyday decision making and the influence they had across so many parts of the business. I also identified a need for people who could bridge the gap between R&D and marketing—which influenced my decision to pursue an MBA. After receiving my MBA, I took roles in traditional CPG marketing organizations to learn the ropes.

  4. Why Invitrogen?
    Life Technologies is an opportunity to use all of my skills, both as a scientist and a marketing manager, for a market-leading company.

  5. What is your favorite part of the job?
    Being at the center of the daily operations of a major influence in the life science research community that is continuously innovating and developing new products. Because of our influence, we are a big target for the competition, which means new challenges are always waiting around the corner.

  6. What keeps you up at night?
    I actually sleep pretty well, but in the back of my mind I want to know more about what the competition is developing and how customers will perceive it.

  7. What is next in store for you?
    I'm excited to help bring really cool new technologies to our customers. We have some new tools that will help customers do breakthrough science, make their research easier and hopefully get them excited about Life Technologies.

  8. What is something most of your colleagues don’t know about you?
    I am an unstoppable, apocalyptic force of nature on the ice in NHL94
    (AKA “The Greatest Video Game EVER”).

Contest

October competition: Did you dress up for Halloween? Send us a picture, and we’ll send an iPod touch® device to the person wearing the best costume. To enter, simply send picture to benchtopicscontest@lifetech.com by November 5, 2010.

* No purchase necessary to enter or win. To be eligible, you must be a legal U.S. (excluding Puerto Rico) or Canadian (excluding Quebec) resident, age 21 and older and employed in a capacity where you perform life sciences research in a private, educational, public, or government research facility or institution. To participate, you must submit picture of yourself in a Halloween costume to benchtopicscontest@lifetech.com, no later than November 5, 2010. Only one submission per person. The eligible entrant who submitted the best picture as solely determined by a panel of judges composed of Invitrogen’s employees (a “Winner”) will win an iPod® touch device. The decision of the panel is final. Void where prohibited, restricted, licensed or otherwise limited by law. The Winner must warrant that they are not prohibited by employment, contract, law or otherwise, including the policies and practices of their employer, from entering or accepting a prize from Invitrogen, the contest sponsor. The Winner will be notified on or about November 7, 2010 by certified mail, e-mail, or telephone. Apple, Inc. is not a sponsor of this contest.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.