GFP-Trap Referral Program

This week we are starting a GFP-Trap referral program. Tell your colleagues about GFP-Trap (or RFP-Trap) and for every two who order you will receive 0.5ml of GFP-trap for free. For more details about this offer visit our promotions page. GFP-Trap is a high quality, single domain GFP-binding protein coupled to a monovalent matrix for biochemical studies (Pulldown, Immunoprecipitation, Co-IP, Western Blot, Mass spec or ChIP) of your fluorescent fusion proteins and their interacting partners. GFP-Trap works with GFP, eGFP, eYFP or Venus (any jellyfish/Aequora victoria derived fluorescent proteins) and GFP-tagged fusion proteins.

We also now have samples of GFP-Trap, RFP-Trap, GFP-multiTrap, and both GFP and RFP-booster for evaluation. To request a sample give us a call at 800-991-7624, or email oligo@allelebiotech.com.

Our New Website is Complete, 10% off this week

We recently launched our new website, and to celebrate the launch, we’ll be offering a discount of 10 % on all our products and services from July 18th to July 22nd! Come explore a wide variety of products that Allele Biotech has to offer from viral expression to fluorescent proteins. Examples of services offered include custom lentiviral, retroviral, and baculoviral packaging along with cell production and cell line development. This is our effort to enhance your online shopping experience through our improved shopping cart system. Our mission remains the same; to increase accessibility to innovative molecular biology research tools by offering cutting edge products at a reasonable cost. Please visit http://www.allelebiotech.com and use the code NEWSITE to redeem the offer. We thank you for your support!
For more details click here

Tags: Fluorescent proteins, iPS, Online Shopping, RNAi, viral packaging

Single Molecule Pulldown can be optimized with Allele’s Fluorescent Proteins

In the recent Nature paper, “Probing cellular protein complexes using single-molecule pull-down”, published May 26, 2011, researchers at the University of Illinois at Urbana-Champaign outline their findings for a new method for visualizing protein complexes through a single-molecule immunoassay which combines an antigen capturing chip and TIRF Microscopy. The SiMPull method captures protein complexes and the captured complexes are then visualized with fluorescent dyes or fluorescent protein tags. This is accomplished by using a microscope slide covered with biotinylated polyethylene glycol (PEG) and streptavidin bound to biotinylated antibodies. For single molecule visualization, multicolor labeling provides differentiation of subcomplexes and configurations.

The study includes two validation experiments, where study team members tagged their chosen complexes with YFP, in order to estimate YFP concentration after pulldown and subsequent imaging. They were then able to determine stoichiometric information in human kidney cells, from the isolated monomeric or dimeric YFPs, which exhibited the one and two-step decay responses.

Additionally, in another validation experiment, team members chose to use protein kinase A (PKA) because it’s two catalytic and two regulatory subunits separate in the presence of cAMP. This was accomplished by labeling the catalytic subunits of protein kinase A with YFP and the regulatory subunits with mCherry fluorescent protein. They then used a two-color SiMPull to pull down PKA. After pull-down they imaged the PKA in the presence of cAMP and without cAMP present. The YFP and mCherry signals fluoresced together, demonstrating that the catalytic and regulatory subunits were still attached to eachother. The YFP and mCherry signals did not correlate in the presense of cAMP, reaffirming the fact that the two subunits disassociate in the presence of cAMP.

Unlike other single-molecule pulldown techniques, the SiMPull does not require purified proteins. It also only requires about 10 cells of sample for protein pull-down and analysis, while traditional Western Blots require about 5000 cells. Moreover, This two-color SiMPull method could be further optimized yielding higher resolution overlay when used in combination with Allele’s mTFP1 and LanYFP, the brightest fluorescent proteins on the market.
The full article can be found at http://www.nature.com/nature/journal/v473/n7348/full/nature10016.html

New Product of the Week: High quality Anti-FLAG Monoclonal Antibody for detection or pulldown, ABP-MAB-DT006, $219/100ug.

Promotion of the Week: save 5% on all of our pre-packaged viruses. Our pre-packaged viruses are all titered at 10^8th or higher, and packaged in 5 tubes for your convenience. To redeem this offer email the code PREPACK to abbashussain@allelebiotech.com.

Tags: cAMP, Jain, LanYFP, mCherry, mTFP1, PKA, SiMPull, TIRF Microscopy, YFP

mRNA Transfection for Better Transgene Expression

Different approaches have been developed to over-express or ectopically express a protein in cells: peptide or full length recombinant protein transfer, viral gene transfer, non-viral DNA transfer and non-viral mRNA transfer.

1) Peptide transfection can be efficient, yet it is limited to only a small part of the protein, limiting the functional potential. Protein transfection is not consistent enough so far, because of the complicated properties of different proteins. Allele Biotech has tested dozens of proteins with several proprietary reagents, leader peptides, etc. but we have decided not to carry a protein transfection product line due to its instability. Furthermore, protein production is an expensive and laborious process.

2) Viral gene transfer is very effective, such as the HIV-based lentivirus or MMLV-based retrovirus, adenovirus, adeno-like virus or baculovirus, etc. However, the potent side-effect will still need to be considered for certain applications, especially involving clinical studies. Nevertheless, as research tools, viral gene transfer is still a highly preferred method. Allele Biotech has been providing the most effective platform for both MMLV-based and HIV-1-based retrovirus packaging. Check out our product website for details.

3) Non-viral DNA transfer is the most widely used transgene method in the biological research community, due to the simplicity of the procedure. There are many commercial kits on the market. However, the low efficiency for transfecting most primary cells significantly limits their use. In recent years, several leading biotech companies have developed various electroporation systems to improve the transfection efficiency and cell viability; although these improvements help with getting DNA inside the cytoplasm, they hardly help transport it into nucleus where DNA is transcribed.

4) Non-viral mRNA transfer has been around for a long time, but it is not widely used. It made a big splash recently through its use for iPSCs reprogramming. IPSCs factor mRNAs greatly improved the iPSCs induction efficiency and completely avoided the viral integration. Other well-known examples of mRNA transfection include loading special cancer antigens or HIV antigens to dendritic cells (DCs) in vitro for personal immunotherapy. PSA antigen expressing DCs transfected by mRNA has moved on to Phrase I Clinical Trials for this purpose.

New Product of the Week: 3C protease immobilized on beads for GST, His tag removal, email oligo@allelebiotech.com for details.

Promotion of the week: 10% off on all fluorescent proteins. To redeem, email oligo@allelebiotech.com along with PROMO code: JELLYFISH

Tags: 5-methylcytidine (5mC), B18R, in vitro transcription, interferon, iPSC factor mRNA, iPSC Warren, modified mRNA, mRNA iPSC, mRNA transfection, mRNA transfer, mRNA transgene, pseudouridine (psi), RiPSC, T7 RNA polymerase, VPA

Recombinase-Mediated Cassette Exchange (RMCE) and Integrase Swappable in vivo Targeting Element (InSITE)

Genetic switches that can turn on expression of genes of interest in specific tissues or time frames have been used in dissecting biological processes. For example, the yeast-derived transcription factor Gal4 and its upstream activating sequence (UAS) are widely adopted in Drosophila for directing gene expressions.

RMCE was designed to replace existing gene control cassette such as UAS-Gal4 with others through the functions of recombinases, such as Cre, Flp, ?C31. Multiple recombination systems were recently combined to form a flexible enhancer trap, InSITE, for exchanging Gal4 with any other sequence (1). The purpose is to use different existing fly lines that express recombinases in various types of cells.

Related to this topic, UAS-Gal4 was combined with “intra-cassette” recombination for selecting one of the three fluorescent proteins (FPs) in tandem in order to label individual neurons in a setup called “Brainbow”, which was first created in mouse, and recently in flies (2). We expect that InSITE and Drosophila Brainbow would be put together for even broader use of different fly lines and in more cell lineage studies.

One technical barrier for effective use of these systems is the lack of high quality FPs that could provide better brightness and narrower emission spectrum than those currently available. For this reason, the above referenced fly Brainbow research relied heavily on using antibodies against tags fused to the FPs, rendering live cell imaging unobtainable in most cases.

A number of the new FPs in Allele Biotech’s pipeline will be introduced to the field soon, such as the Lancelet YFP, that is ~10X brighter than EGFP, with a very narrow emission bandwidth.

(1) Gahl et Al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1561.html
(2) Hampel et al. 2011, http://www.nature.com/nmeth/journal/v8/n3/full/nmeth.1566.html

New Product of the Week 022811-030611:

NCI certified Real Time PCR primer set, coming soon to online ordering, email oligo@allelebiotech.com for details.

Promotion of the week 022811-030611:

Save 10% on all Protein Extraction Reagents. Email brianahasey@allelebiotech.com with offer code: proteinextract.

Tags: ?C31, Brainbow, cre, Drosophila Brainbow, Flp, InSITE, RMCE, UAS-Gal4