Allele’s pallet of the super star fluorescent proteins

“Photoblog”–just some fun pictures from our notebooks.

The brightest cyan, green fluorescent proteins, and the brightest ever FP in LanYFP!

Ain't they pretty?

These fluorescent proteins are representatives of the growing family or high quality, new generation FPs engineered to enable experiment previously deemed impossible.

Cells infected with lentivirus carrying mWasabi. Lentivirus carrying LanYFP will make most cells much more brighter than this.

The mWasabi is stimulating

The brightest green fluorescent protein with excellent photostability, carried on 10e8 TU/ml high titer lentivirus.

The LanFPs express well in bacteria.

The LanFPs express well in bacteria

Project planning is under way to test the cytotoxicity of lanFPs in different mammalian cell lines and in vivo with a focus on neurons.

The FPs fold so strongly that they fluorescence even in SDS-PAGE.

Can you see the FP bands in the SDS PAGE?

FPs in SDS PAGE–a closer look

Can you see them now?

FPs in gel cassette over UV lights

Invincible FPs

FPs in gel cassette under blue LED

Fluorescence in SDS page under blue LED

The purified FPs can be used as “real time” protein markers.

New Product of the Week 07/26/10-08/01/10: pCHAC-mWasabi-C for expressing mWasabi fusion through retroviral vectors.

Promotion of the Week 07/26/10-08/01/10: Get 3′ TAMRA & BHQ oligo mods for $45 ea & 3′ Dabcyl mod for $20 50 nmol syn scale only/while supplies last- use dbtkrm0726

Tags: Fluorescent proteins, FP, FPs, FRET, GFP, lab note, lentivirus, mCherry, mTFP1, mWasabi, photos, pictures

17 Papers Using GFP-Trap, 12 Since 2009

1. MacKay C, Déclais AC, Lundin C et al. (2010). Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142:65-76.

2. Babiano R, de la Cruz J. (2010). Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae. Nucleic Acids Res 2010 Apr 14.

3. Fulcher AJ, Dias MM, Jans DA. (2010). Binding of p110 retinoblastoma protein inhibits nuclear import of simian virus SV40 large tumor antigen. J Biol Chem. 285:17744-53.

4. Taniue K, Nishida A, Hamada F et al. (2010). Sunspot, a link between Wingless signaling and endoreplication in Drosophila. Development. 137:1755-64.

5. Rottach A, Frauer C, Pichler G et al. (2010). The multi-domain protein Np95 connects DNA methylation and histone modification. Nucleic Acids Res. 38:1796-804.

6. Boulon S, Ahmad Y, Trinkle-Mulcahy L et al. (2010). Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners. Mol Cell Proteomics. 9:861-79.

7. Schornack S, Fuchs R, Huitema E et al. (2009). Protein mislocalization in plant cells using a GFP-binding chromobody. Plant J. 60:744-54.

8. Fellinger K, Bultmann S, Rothbauer U et al. (2009). Np95 interacts with de novo DNA methyltransferases, Dnmt3a and Dnmt3b, and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells. EMBO Rep. 10:1259-64.

9. Muñoz IM, Hain K, Déclais AC et al. (2009). Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair. Mol Cell. 35:116-27.

10. Webby CJ, Wolf A, et al. (2009). Jmjd6 Catalyses Lysyl-Hydroxylation of U2AF65, a Protein Associated with RNA Splicing. Science. 325:90-93.

11. Rogowski K et al. (2009). Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell. 137: 1076-87.

12. Frauer C, Leonhardt H, (2009) A versatile non-radioactive assay for DNA methyltransferase activity and DNA binding. Nucleic Acid Res. 35: 5402-5409.

13. Trinkle-Mulcahy L et al., (2008) Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes. J Cell Biol. 183: s223-39.

14. Rothbauer U, Leonhardt H, (2008) Connecting Biochemistry and Cell Biology with Nanobodies. Zellbiologie aktuell 34: 9-12.

15. Rothbauer U et al., (2008) A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol Cell Proteomics 7: 282-289.

16. Agarwal N et al., (2007) MeCP2 interacts with HP1 and modulates its heterochromatin association during myogenic differentiation. Nucleic Acid Res.35: 5402-5409.

17. Rothbauer U et al., (2006) Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat Methods 3: 887-889.

New Product of the Week 071910-072510: Cre Reporter Cell Line: LoxP-RFP Human Fibroblast, perfect to test our Cre-2A-GFP lentivirus, when cre works, the cell change from red to green.

Promotion of the Week 071910-072510: High Quality dNTP Mix, 10mM, 5 ml, $409 this week $309. Hurry, email to oligo@allelebiotech.com or fax 858-587-6692 by Sunday to save $100 on your lab budget.

Tags: anti-GFP, chromatin immunoprecipitation (ChIP), coIP, GFP, immunoprecipitation, nAb: Camelid Antibodies, Nanobodies, VHH, pull down

Monomeric Photoconvertible Fluorescent Protein for Imaging of Dynamic Protein Localization

Allele Biotech has just made a news announcement indicating that researchers from Dr. Campbell’s lab at the University of Alberta, Canada, and scientists at Allele Biotech Drs. Nathan Shaner and Jiwu Wang published a paper in the Journal of Molecular Biology on July 5th introducing a new photoconvertible fluorescent protein mClavGR.

The use of green-to-red photoconvertible fluorescent proteins (FPs) enables researchers to highlight a subcellular population of a fusion protein of interest and image its dynamics in live cells. In an effort to enrich the arsenal of photoconvertible FPs and overcome the limitations imposed by the oligomeric structure of the natural photoconvertible FPs, we designed and optimized a new monomeric photoconvertible FP. Furthermore, we have exploited mClavGR2 to determine the diffusion kinetics of the membrane protein intercellular adhesion molecule 1 (ICAM-1) both when the membrane is in contact with a T lymphocyte expressing leukocyte function-associated antigen 1 (LFA-1) and when it is not. These experiments clearly establish that mClavGR2 is well suited for rapid photoconversion of protein sub-populations and subsequent tracking of dynamic changes in localization in living cells.

Compared with previously available photoconvertible FPs, mClavGR2 has much improved photostability of the red state under confocal illumination conditions, 3644 over mEOS2’s 2700 and Dendra2’s 2420. Most notable among other advantages of mClavGR2 is its monomeric structure, its highly optimized and relatively rapid folding efficiency, and its high photoconversion effi ciency due to the high pKa of the green state. Its brightness in both the green and the red states is similar to the popular mCherry.

In regard to monomeric state, the monomeric variant of EosFP, known as mEos, was created through the introduction of two point mutations that disrupted the protein-protein interfaces of the tetrameric species. Expression of mEos at temperatures of greater than 30 °C is problematic, but an effectively monomeric tandem dimer variant does express well at 37 °C. mEos2 has been reported to retain some propensity for dimer formation.

We anticipate that this new addition to the toolbox of engineered FPs will be of great utility in imaging of fast protein dynamics in live cells. Experiments to determine whether the advantages of mClavGR2 translate to improved performance in super-resolution imaging applications have been initiated.

Hiofan Hoi(a), Nathan C. Shaner(b), Michael W. Davidson(c), Christopher W. Cairo(a), d, Jiwu Wang(b) and Robert E. Campbell(a)
a University of Alberta, Department of Chemistry, Edmonton, Alberta, Canada T6G 2G2
b Allele Biotechnology, 9924 Mesa Rim Road, San Diego, California 92121
c National High Magnetic Field Laboratory and Department of Biological Science, The Florida State University, 1800 E. Paul Dirac Dr., Tallahassee, Florida 32310
d Alberta Ingenuity Centre for Carbohydrate Science
Received 20 February 2010; revised 15 June 2010; accepted 25 June 2010. Available online 5 July 2010.

New Product of the Week 070510-071110: mClavGR greeen-to-red photoconvertible fluorescent protein, catalogue number to be created

Promotion of the Week 070510-071110: Purified lanYFP, bright even in SDS-PAGE gel WITHOUT dye or excitation, great for in gel marker.

Tags: Dendra2, dynamic imaging, FP, FPs, mCherry, mEOS, mKirGR, PALM, photoconvertible, photoswitchable, STORM

Brightest Ever Fluorescent Protein

LanYFP, identified from lancelet (also known as amphioxus, e.g. Branchiostoma floridae), has been found to have the following properties:

Excitation 513nm
Emission 524nm
Quantum yield 0.95
Extinction coefficient 150,000
pKa ~3.5
Salt insensitive 0-500mM NaCl

LanYFP has a brightness of 143! For comparison, the brightness of the previously known brightest FPs is 95 for tdTomato, and 34 for commonly used EGFP.

Allele already has been exclusively providing the brightest cyan FP in mTFP1 (brightness of 54); and the brightest green FP in mWasabi (brightness of 56). The confirmation of LanYFP as the brightest ever FP is a major milestone of Allele’s research and development efforts in the fluorescent protein field. We are currently monomerizing LanYFP and another lancelet protein, LanRFP. Once completed, the new proteins should definitely be the FPs of choice for in vivo imaging and FRET with unprecedented utilities.

Promotion of the week 062010-061610: Validated Rex1 Promoter Reporter Lentiviral Particles-1 Vial for $149.00 (ABP-SC-RREX2R1). Save $59 if place an order this week! http://www.allelebiotech.com/shopcart/index.php?c=200&sc=34

New product of the week, recombinant mTFP1, mWasabi, LanYFP, LanRFP, $159 for 125 ug, compare price for 100ug vs 125ug in other companies’ offers, you will know that you are getting a good deal from Allele.

Tags: brightest FP, fluorescent protein, Fluorescent proteins, FP, FP brightness, FPs, Lancelet, monomer FP, monomerization, mTFP1, mWasabi, RFP, YFP

Developing Cell-Based Assays

Cascaded protein interactions form the foundation of all signaling pathways, many of which are involved in multiple human diseases. These interactions are sensitively and precisely regulated by various post-translation modifications such as phosphorylation, acetylation, ubiquitination, etc. Many action points of the protein modifications have been targeted during drug development. From a survey we have conducted on assays aimed at these targets, we found that most of the assays are based on the enzymatic reactions, e.g. phosphorylation-specific ELISA, and chemically modified FRET, which require pre-assembled reagent kits which are hard to apply to different targets and different cell models.

Fluorescent Protein based FRET might be the optimal choice to develop a versatile cell-based assay. Since signaling pathways rely on hierarchical protein-protein interactions, the most direct and precise way to study cell signaling pathways would be to detect the interactions between a target protein and its immediate downstream protein. Furthermore, different upstream signals can activate the same set of target proteins in different post-modification patterns, resulting in specific activation of downstream responding factors. These signal flows may be individually monitored by using FRET based assay redesigned and validated for each downstream pathway.

Allele’s scientists can develop cell-based assays with in-depth understanding of protein interactions within the context of human genome, such as the SH2, SH3 and PTB domains in tyrosine kinase signaling, the F-box, BTB-box, SOCS, WDR, and LRR domains in the ubiquitin proteasome system, etc. Additionally, Allele’s cell-based assays can be carried on world’s most powerful lentivirus packaging platform, suitable for virtually all different cell lines and primary cells.

New Product of the Week 061410 to 062010: Rat monoclonal antibody against GFP, strong signal for GFP labeling

Promotion of the Week 061410 to 062010: Mouse LIF-bFGF expressing feeder cells for stem cell culture (ABP-SC-BLIFM05), one vial free to go with any iPS lentivirus/retrovirus kit.

Tags: BTB-box, cell based assays, F-box, FRET, LRR domain, pathway, signaling pathway, SOCS, WDR domain