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

Making a difference for those in need

This month 4 major pharmaceutical companies, GlaxoSmithKline, Merck, Johnson & Johnson and Sanofi-Aventis; all agreed to lower the cost of critical vaccines for developing countries. They have all done this as a part of the international vaccine alliance GAVI. The companies have severely slashed the prices of vaccines for diseases like rotavirus, a disease that isn’t prevalent in developed countries but causes more than half a million deaths per year. The model GAVI uses is one where vaccine costs are drastically lowered in developing countries, and this cost is offset by raised vaccine prices in developed countries.

GlaxoSmithKline has also reported that they are very close to developing an anti-malaria vaccine, which would be the first of its kind. This clearly shows a dissent from common pharmaceutical business practice, since Malaria is virtually wiped out in most developed countries. GSK has no hope of recouping costs for this vaccine by having patients in developed countries pay a premium for vaccination; but this has not deterred their efforts. Rather they have pledged to make on a 5% profit of the sale of the vaccine which will go toward future anti-malaria drug research.

Pharmaceutical companies are often viewed in a negative light for their practice of charging a premium for new drugs. However, the research, development, trials, and further clinical trials required to bring a drug or vaccine to market are all very costly, somewhat justifying a new drug’s high cost. Unfortunately this means there is no market for new drugs to combat diseases in developing countries as they cannot afford to compensate drug companies accordingly for their development costs. This is the key flaw in GAVI’s model, so it is great to see GSK is unhindered by this fact.

Everyday people who work in the biotech field strive to make a difference and help humanity through their research. Through the work of organizations like GAVI this research can ideally be utilized by all, and not just by those who can afford it.

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Tags: GlaxoSmithKline, healthcare, Johnson & Johnson, Merck, pour countries, Sanofi-Aventis, vaccines

Generate mouse and human iPS cells with transfected mature miRNAs

In last week’s blog we discussed generation of induced pluripotent stem cells (iPSCs) with miRNAs expressed from lentivirus. To take it a step further, synthetic, mature miRNAs can be used to avoid the use of viral vectors. Sure enough, Miyoshi et al. published a paper online a few days ago showing that by transfecting 6 miRNAs at 48 hour intervals, they were able to create iPSCs from mouse and human somatic cells. The efficiency is comparable to retrovirus-mediated OSKM factor over-expression (Yoshida et al.), and therefore lower than lentivirus-mediated miR302/369 expression (Anokye-Danso et al.).

In the study of using mature miRNA for obtaining iPSCs, the researchers transfected miRNAs mir200c, mir302s and mir-369 into tissue cultured cells and achieved reprogramming results. Interestingly, only mir302s are common between this study and that with lentivirus-mediated miRNAs by Anokye-Danso et al. There is no current explanation as to why mir-367, which was shown to be required by Anokye-Danso et al., did not seem to be needed in the mature miRNA transfection experiments. Perhaps a level of redundancy among miRNAs, combined with their broad target range and relatively low specificity, allow some of the miRNAs to be interchangeable when used for reprogramming.

Finally, neither of these two recent miRNA-iPSCs works was the first to demonstrate that miRNAs can initiate or facilitate reprogramming. As early as 2008, Lin et al. showed that mir302s could induce pluripotency in a dose-dependent manner by using tet-induced lentivirus expression. They further illustrated that the underlying mechanism is likely through mir302s’ regulation of epigenetic regulators AOFs and other similar factors.

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Tags: Anokye-Danso, iPS cells, iPSCs, mir-367, mir-369, mir200c, mir302s, Miyoshi et al., no foot print iPSCs, pluripotency