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

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

Use of Fluorescent Protein in Studying Protein Half-Life

How long a protein remains in cell and at what equilibrium level depends on several factors: 1) how fast it is translated; 2) how fast it is degraded; 3) how much dilution by cell division affects its balance. A good method for tracking protein degradation requires live cell measurement methods that show high resolution because the changes may be small and gradual; and that do not interfere with cellular processes. One simple method was recently described in Science by Eden et al. that relies on bleaching fluorescent protein (FP) tagged to cellular protein of interest.

To track protein half-life, only a small fraction of FP is bleached with a pulse of light that would irreversibly damage the chromophore of the FP. This treatment, called bleach chase, would produce a population of proteins that are non-fluorescent and cannot be replenished. By comparing the fluorescence of this population and the control, unbleached population, it is possible to determine the half-life of the fused proteins using equation T1/2=ln(2)/a, where a is the slope of decay of the difference between bleached and unbleached protein fluorescence on a semilogarithmic plot. (This part is recited from AlleleNews)

Conversely, instead of photobleaching a FP to create a protein population, a fluorescent signal can be created and chased by photoactivating a photoactivable FP that is fused to a cellular protein under study. Plachta et al. published in a recent issue of Nature Cell Biology that by following the half-life, or kinetics of pluripotency-related transcription factor Oct4, cell fates are predicted in early embryo development.

In fact, there is a third method, perhaps soon to be published, that a photoconvertible FP can be used for tracking fusion protein half life. By using a photoconvertible FP, such as mClavGR (already offered by Allele), a fluorescent protein population can be created as in the aforementioned studies; but unlike bleaching or photoactivating, photoconversion keeps both populations (converted and unconverted, green or red in the case of mClavGR) present. This way all readings can be internally controlled to compensate for factors not directly related to protein metabolism per se, such as cell death, equipment variation, etc.

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Tags: bleach chase, cell fate, mClavGR, Oct4 kinetics, photoactivable FP, photoconvertible FP, protein half life, pulse chase, YFP

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.

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

Lentivirus-lanYFP Give Away

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Tags: EGFP, Fluorescent proteins, GFP, Lancelet, lentivirus, mTFP1, mWasabi, pre-validated virus, YFP