Resolution-enhanced native acidic gel electrophoresis: A method for resolving, sizing, and quantifying prion protein oligomers

Ladner, Carol L.; Wishart, David S.

doi:10.1016/j.ab.2012.04.005

Cited by 19 publications

(38 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This differs from PrP β oligomers formed by urea (Fig. 6) or acid, 33 which are stable under electrophoresis conditions. These findings may have significant consequences for laboratories using recombinant prion proteins in PMCA or in vitro conversion assays and for laboratories involved with animal prion transmission experiments.…”

Section: Discussionmentioning

confidence: 77%

“…33 With RENAGE it has been previously shown that the PrP β oligomers formed via urea/salt conversion consist of a distribution of heptamers to dodecamers. 33 This size distribution of medium-sized oligomers is seen in Figure 6. In contrast, LPS-converted prion protein (PrP β ) are characterized by very large aggregates that migrate just a few millimeters into the stacking gel.…”

Section: Size Comparison Of Prp β Formed By Conversion and Fibril Promentioning

confidence: 99%

“…33 Briefly, converted prion protein (8 μg) was loaded onto an 8% RENAGE gel at pH 4.3 with a 3% stacking gel at pH 5.2. The gels were pre-run for 20 min prior to loading protein samples.…”

Section: Nmr Analysismentioning

confidence: 99%

See 2 more Smart Citations

Lipopolysaccharide induced conversion of recombinant prion protein

Saleem

Bjorndahl

Ladner

et al. 2014

Prion

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 77%

Section: Size Comparison Of Prp β Formed By Conversion and Fibril Promentioning

confidence: 99%

See 1 more Smart Citation

Lipopolysaccharide induced conversion of recombinant prion protein

Saleem

Bjorndahl

Ladner

et al. 2014

Prion

View full text Add to dashboard Cite

“…Truncated recombinant prion proteins from Syrian hamster, mouse and white-tailed deer (cervid) constructs (recShPrP 90–232 , recMoPrP 90–231 , recMoPrP 120–231 and recCePrP 94–233 ) with His6x-tags were expressed in E. coli and purified as previously described [9], [22]. In addition, a full-length recMoPrP 23–231 construct was generated similarly by inserting MoPrP 23–231 into a pET15b expression plasmid containing an N-terminal fusion tag attached to a His6x tag, a thrombin cleavage site and an enterokinase cleavage site (MGSSHHHHHHSSGLVPRGSHMDDDD).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, a full-length recMoPrP 23–231 construct was generated similarly by inserting MoPrP 23–231 into a pET15b expression plasmid containing an N-terminal fusion tag attached to a His6x tag, a thrombin cleavage site and an enterokinase cleavage site (MGSSHHHHHHSSGLVPRGSHMDDDD). All constructs were purified on Ni-NTA (Qiagen Canada, Toronto, Canada) as previously described [22] and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, USA) added to eluted PrP fractions at a dilution of between 100X and 25X, from a tablet dissolved in 1 mL of water. In addition, for the full-length MoPrP 23–231 , 1 mM EDTA was added to the eluted PrP fractions.…”

Section: Methodsmentioning

confidence: 99%

Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils

2014

Self Cite

View full text Add to dashboard Cite

The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).

show abstract

Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single‐Molecule Diffusivity in the Membrane of Living Cells

et al. 2015

View full text Add to dashboard Cite

We present as ingle-molecule diffusional-mobilityshift assay(smDIMSA) for analyzing the interactions between membrane and water-soluble proteins in the crowded membrane of living cells.Wefound that ligand-receptor interactions decreased the diffusional mobility of ErbB receptors and badrenergic receptors,asdetermined by single-particle tracking with super-resolution microscopy. The shift in diffusional mobility was sensitive to the size of the water-soluble binders that ranged from af ew tens of kilodaltons to several hundred kilodaltons.This technique was used to quantitatively analyze the dissociation constant and the cooperativity of antibody interactions with the epidermal growth factor receptor and its mutants.s mDIMSA enables the quantitative investigation of previously undetected ligand-receptor interactions in the intact membrane of living cells on the basis of the diffusivity of single-molecule membrane proteins without ligand labeling.Onthe surface of ap lasma membrane,t he interactions of membrane proteins with ligands are crucial to the communication between the intra-and extracellular environments. [1] Va rious types of ligand-receptor interactions occur in the membrane of living cells,yet many of these interactions have not been elucidated.[2] Because the crowded and heterogeneous cellular membrane generates unique biochemical conditions for biomolecular reactions, [3] membrane-protein interactions should be investigated in the intact membrane of living cells if their genuine biochemical properties are to be understood.

show abstract

Resolution-enhanced native acidic gel electrophoresis: A method for resolving, sizing, and quantifying prion protein oligomers

Cited by 19 publications

References 34 publications

Lipopolysaccharide induced conversion of recombinant prion protein

Lipopolysaccharide induced conversion of recombinant prion protein

Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils

Analysis of Interactions between the Epidermal Growth Factor Receptor and Soluble Ligands on the Basis of Single‐Molecule Diffusivity in the Membrane of Living Cells

Contact Info

Product

Resources

About