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Aggregation kinetics of <t>α-synuclein</t> in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.
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Images

1) Product Images from "α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay"

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

Journal: Bio-protocol

doi: 10.21769/BioProtoc.2941

Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.
Figure Legend Snippet: Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.

Techniques Used: Size-exclusion Chromatography, Fluorescence

Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).
Figure Legend Snippet: Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).

Techniques Used: Fluorescence, Concentration Assay

2) Product Images from "α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay"

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

Journal: Bio-protocol

doi: 10.21769/BioProtoc.2941

Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.
Figure Legend Snippet: Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.

Techniques Used: Size-exclusion Chromatography, Fluorescence

Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).
Figure Legend Snippet: Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).

Techniques Used: Fluorescence, Concentration Assay

3) Product Images from "Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations"

Article Title: Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations

Journal: Biochemistry

doi: 10.1021/acs.biochem.9b00215

Aggregation kinetics of α-synuclein (0.75 mg/ml, 50 μM, circle), tau (1.5 mg/ml, 30 μM, ×), and mixtures of α-synuclein and tau (50 μM and 15 μM, 1:0.3, square; 50 μM and 30 μM, 1:0.6, triangle). Optical density (O. D.) was measured at 400 nm for turbidity of the samples. The accelerated aggregation kinetics was also observed at different protein concentrations (70 μM and 20 μM for α-synuclein and tau, respectively).
Figure Legend Snippet: Aggregation kinetics of α-synuclein (0.75 mg/ml, 50 μM, circle), tau (1.5 mg/ml, 30 μM, ×), and mixtures of α-synuclein and tau (50 μM and 15 μM, 1:0.3, square; 50 μM and 30 μM, 1:0.6, triangle). Optical density (O. D.) was measured at 400 nm for turbidity of the samples. The accelerated aggregation kinetics was also observed at different protein concentrations (70 μM and 20 μM for α-synuclein and tau, respectively).

Techniques Used:

CD spectra of pure tau (10 μM) (a), a mixture of tau (10 μM) and α-synuclein monomer (1 μM) (b), and a mixture of tau (10 μM) and α-synuclein filaments (1 μM, monomer concentration) (c) acquired at different incubation times. The CD signal of the mixtures decreased due to the precipitation of the protein aggregates.
Figure Legend Snippet: CD spectra of pure tau (10 μM) (a), a mixture of tau (10 μM) and α-synuclein monomer (1 μM) (b), and a mixture of tau (10 μM) and α-synuclein filaments (1 μM, monomer concentration) (c) acquired at different incubation times. The CD signal of the mixtures decreased due to the precipitation of the protein aggregates.

Techniques Used: Concentration Assay, Incubation

CD spectra of pure α-synuclein (10 μM) (a), pure tau (3 μM) (b), and a mixture of α-synuclein (10 μM) and tau (3 μM) (c) acquired at different incubation times. The CD signal of the mixture after 5 days of incubation decreased due to the precipitation of the protein aggregates.
Figure Legend Snippet: CD spectra of pure α-synuclein (10 μM) (a), pure tau (3 μM) (b), and a mixture of α-synuclein (10 μM) and tau (3 μM) (c) acquired at different incubation times. The CD signal of the mixture after 5 days of incubation decreased due to the precipitation of the protein aggregates.

Techniques Used: Incubation

(a) Overlaid 1 H/ 15 N HSQC NMR spectra of α-synuclein (70 μM, pH 7.4, black) and tau (140 μM, red) at 15 °C. (b) Weighted sum of the 1 H and 15 N Chemical shift changes of α-synuclein (70 μM) upon addition of tau (140 μM), Δ δ = δ H 2 + 0.2 δ N 2 . (c) Transverse relaxation rates (R 2 and 3D NMR experiments.
Figure Legend Snippet: (a) Overlaid 1 H/ 15 N HSQC NMR spectra of α-synuclein (70 μM, pH 7.4, black) and tau (140 μM, red) at 15 °C. (b) Weighted sum of the 1 H and 15 N Chemical shift changes of α-synuclein (70 μM) upon addition of tau (140 μM), Δ δ = δ H 2 + 0.2 δ N 2 . (c) Transverse relaxation rates (R 2 and 3D NMR experiments.

Techniques Used: Nuclear Magnetic Resonance

TEM images of the pure α-synuclein (210 μM) (a) and mixtures of α-synuclein (70 μM) and tau (20 μM) at a molar of 1:0.3 (b). The pure α-synuclein and mixture of the two proteins were incubated at 37 °C for one week and one day, respectively. (c) 2D 13 C- 13 C correlation solid-state NMR spectra for the α-synuclein filaments formed in the absence (black) and presence (red) of tau.
Figure Legend Snippet: TEM images of the pure α-synuclein (210 μM) (a) and mixtures of α-synuclein (70 μM) and tau (20 μM) at a molar of 1:0.3 (b). The pure α-synuclein and mixture of the two proteins were incubated at 37 °C for one week and one day, respectively. (c) 2D 13 C- 13 C correlation solid-state NMR spectra for the α-synuclein filaments formed in the absence (black) and presence (red) of tau.

Techniques Used: Transmission Electron Microscopy, Incubation, Nuclear Magnetic Resonance

4) Product Images from "Neurodegenerative Disease Proteinopathies Are Connected to Distinct Histone Post-translational Modification Landscapes"

Article Title: Neurodegenerative Disease Proteinopathies Are Connected to Distinct Histone Post-translational Modification Landscapes

Journal: ACS chemical neuroscience

doi: 10.1021/acschemneuro.7b00297

FUS and α -synuclein proteinopathies are associated with changes in histone H2B phosphorylation levels. Shown are representative immunoblots displaying the levels of H2BT129ph for (a) TDP-43 and FUS and (b) α -synuclein yeast proteinopathy models. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n ≥ 3) for each modification. *, p
Figure Legend Snippet: FUS and α -synuclein proteinopathies are associated with changes in histone H2B phosphorylation levels. Shown are representative immunoblots displaying the levels of H2BT129ph for (a) TDP-43 and FUS and (b) α -synuclein yeast proteinopathy models. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n ≥ 3) for each modification. *, p

Techniques Used: Western Blot, Quantitation Assay, Modification

α -Synuclein and TDP-43 proteinopathy models display decreases in histone H3 di- and trimethylation, respectively, on lysine 36. Shown are representative immunoblots displaying the levels of (a, c) H3K36me2 and (b, d) H3K36me3 for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM ( n = 3–7) for each modification. **, p
Figure Legend Snippet: α -Synuclein and TDP-43 proteinopathy models display decreases in histone H3 di- and trimethylation, respectively, on lysine 36. Shown are representative immunoblots displaying the levels of (a, c) H3K36me2 and (b, d) H3K36me3 for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM ( n = 3–7) for each modification. **, p

Techniques Used: Western Blot, Quantitation Assay, Modification

FUS overexpression correlates with changes in phosphorylation on serine 10 and acetylation on lysine 14 and lysine 56 of histone H3. Shown are representative immunoblots displaying the levels of (a, d) H3S10ph, (b, e) H3K14ac, and (c, f) H3K56ac for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n = 3–6) for each modification. **, p
Figure Legend Snippet: FUS overexpression correlates with changes in phosphorylation on serine 10 and acetylation on lysine 14 and lysine 56 of histone H3. Shown are representative immunoblots displaying the levels of (a, d) H3S10ph, (b, e) H3K14ac, and (c, f) H3K56ac for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n = 3–6) for each modification. **, p

Techniques Used: Over Expression, Western Blot, Quantitation Assay, Modification

FUS and TDP-43 overexpression correlate with changes in arginine dimethylation and lysine acetylation of histone H4. Shown are representative immunoblots displaying the levels of (a, d) H4R3me2, (b, e) H4K12ac, and (c, f) H4K16ac for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n = 3–6) for each modification. *, p
Figure Legend Snippet: FUS and TDP-43 overexpression correlate with changes in arginine dimethylation and lysine acetylation of histone H4. Shown are representative immunoblots displaying the levels of (a, d) H4R3me2, (b, e) H4K12ac, and (c, f) H4K16ac for TDP-43 and FUS and for α -synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside the blots. All of the graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate +SEM ( n = 3–6) for each modification. *, p

Techniques Used: Over Expression, Western Blot, Quantitation Assay, Modification

RNA levels are decreased in a FUS overexpression ALS model. (a) Total RNA levels in yeast control cells and cells overexpressing TDP-43 and FUS. (b) Total RNA levels in yeast control cells and cells overexpressing  α -synuclein. All of the graphs display the mean fold change in total RNA levels for each group. Error bars indicate +SEM ( n  = 3) for each group. *,  p
Figure Legend Snippet: RNA levels are decreased in a FUS overexpression ALS model. (a) Total RNA levels in yeast control cells and cells overexpressing TDP-43 and FUS. (b) Total RNA levels in yeast control cells and cells overexpressing α -synuclein. All of the graphs display the mean fold change in total RNA levels for each group. Error bars indicate +SEM ( n = 3) for each group. *, p

Techniques Used: Over Expression

Histone modifications are altered in the context of yeast overexpressing TDP-43, FUS, and α -synuclein ( α -Syn). Shown are p values for the ratios of histone H2A, H2B, H3, and H4 PTM abundances in yeast strains expressing TDP-43, FUS, and α -Syn relative to control cells. The scale is based on p values derived from statistical analysis of Western blotting experiments. The p values were calculated using a two-tailed t test with Welch’s modification. Green indicates more statistically significant decreased modification levels, while red indicates more statistically significant increased modification levels.
Figure Legend Snippet: Histone modifications are altered in the context of yeast overexpressing TDP-43, FUS, and α -synuclein ( α -Syn). Shown are p values for the ratios of histone H2A, H2B, H3, and H4 PTM abundances in yeast strains expressing TDP-43, FUS, and α -Syn relative to control cells. The scale is based on p values derived from statistical analysis of Western blotting experiments. The p values were calculated using a two-tailed t test with Welch’s modification. Green indicates more statistically significant decreased modification levels, while red indicates more statistically significant increased modification levels.

Techniques Used: Expressing, Derivative Assay, Western Blot, Two Tailed Test, Modification

Related Articles

High Performance Liquid Chromatography:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Nucleic Acid Electrophoresis:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Transfection:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: .. HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Chromatography:

Article Title: Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations
Article Snippet: The DNA plasmids of α-synuclein (UniProtKB entry ) and tau (UniProtKB entry ) were gifts from Michael J Fox Foundation MJFF (pET21a, Addgene plasmid # 51486) and from Dr. Smet-Nocca (pET15b, Université de Lille, Sciences et Technologies, France), respectively. .. The precipitates were resuspended with deionized water and purified using anion exchange Q column followed by size-exclusion gel-filtration chromatography using a HiLoad 16/600 Superdex 75 column (GE Healthcare, Piscataway, NJ).

Stable Transfection:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: .. HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Fluorescence:

Article Title: α-Synuclein impairs ferritinophagy in the retinal pigment epithelium: Implications for retinal iron dyshomeostasis in Parkinson’s disease
Article Snippet: α-Syn inhibits autophagosomal/lysosomal activity To evaluate whether α-syn interferes with the fusion of autophagosomes with lysosomes, a widely used technique utilizing the pH-sensitive characteristics of GFP and mCherry tagged to the N-terminus of LC3 plasmid was used (Addgene #22418) . .. At the physiological pH of phagophores and autophagosomes, both eGFP (green) and mCherry (red) fluoresce, emitting a yellow fluorescence.

Size-exclusion Chromatography:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Construct:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: .. HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Purification:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Article Title: Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations
Article Snippet: The DNA plasmids of α-synuclein (UniProtKB entry ) and tau (UniProtKB entry ) were gifts from Michael J Fox Foundation MJFF (pET21a, Addgene plasmid # 51486) and from Dr. Smet-Nocca (pET15b, Université de Lille, Sciences et Technologies, France), respectively. .. Wild-type α-synuclein was expressed in BL21 (DE3) E. coli , and was purified as previously described.

Expressing:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: .. HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Article Title: Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations
Article Snippet: Paragraph title: Protein expression. ... The DNA plasmids of α-synuclein (UniProtKB entry ) and tau (UniProtKB entry ) were gifts from Michael J Fox Foundation MJFF (pET21a, Addgene plasmid # 51486) and from Dr. Smet-Nocca (pET15b, Université de Lille, Sciences et Technologies, France), respectively.

Activity Assay:

Article Title: α-Synuclein impairs ferritinophagy in the retinal pigment epithelium: Implications for retinal iron dyshomeostasis in Parkinson’s disease
Article Snippet: .. α-Syn inhibits autophagosomal/lysosomal activity To evaluate whether α-syn interferes with the fusion of autophagosomes with lysosomes, a widely used technique utilizing the pH-sensitive characteristics of GFP and mCherry tagged to the N-terminus of LC3 plasmid was used (Addgene #22418) . .. At the physiological pH of phagophores and autophagosomes, both eGFP (green) and mCherry (red) fluoresce, emitting a yellow fluorescence.

Imaging:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Mass Spectrometry:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Sonication:

Article Title: Tau interacts with the C-terminal region of α-synuclein, promoting formation of toxic aggregates with distinct molecular conformations
Article Snippet: The DNA plasmids of α-synuclein (UniProtKB entry ) and tau (UniProtKB entry ) were gifts from Michael J Fox Foundation MJFF (pET21a, Addgene plasmid # 51486) and from Dr. Smet-Nocca (pET15b, Université de Lille, Sciences et Technologies, France), respectively. .. Briefly, overexpressed proteins in the soluble fraction of the sonicated lysates were extracted using ammonium sulfate precipitation methods (50 %).

Recombinant:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Sterility:

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Thioflavin T, ultrapure grade (AnaSpec, catalog number: AS-88306) Sodium azide ( purum p.a., Sigma-Aldrich, catalog number: 71290) H2 O (Milli-Q ≥ 18.2 MΩ resistivity) Glass beads, Ø 2.85-3.45 mm (Carl Roth, catalog number: A557.1), stored in 70% ethanol to ensure sterility Potassium dihydrogen phosphate ( p.a., AppliChem, catalog number: A1043) Dipotassium hydrogen phosphate, trihydrate ( p.a., Merck, catalog number: 105099) Potassium chloride ( p.a., Carl Roth, catalog number: 6781.1) Hydrochloric acid, p.a.

Plasmid Preparation:

Article Title: Neurodegenerative Disease Proteinopathies Are Connected to Distinct Histone Post-translational Modification Landscapes
Article Snippet: .. Vectors encoding TDP-43, FUS, and α -synuclein (pAG303GAL-TDP-43, pAG303GAL-FUS, and pAG303GAL- α -synu-clein-GFP, respectively) were a gift from A. Gitler., , A pAG303GAL-ccdB vector, which was used as a control, was a gift from Susan Lindquist (Addgene plasmid no. 14133). .. Yeasts were transformed according to standard protocols using poly(ethylene glycol) and lithium acetate.

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: .. HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay
Article Snippet: .. 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry. .. Alternatively, acetic acid gel electrophoresis can be performed ( ).

Article Title: α-Synuclein impairs ferritinophagy in the retinal pigment epithelium: Implications for retinal iron dyshomeostasis in Parkinson’s disease
Article Snippet: .. α-Syn inhibits autophagosomal/lysosomal activity To evaluate whether α-syn interferes with the fusion of autophagosomes with lysosomes, a widely used technique utilizing the pH-sensitive characteristics of GFP and mCherry tagged to the N-terminus of LC3 plasmid was used (Addgene #22418) . .. At the physiological pH of phagophores and autophagosomes, both eGFP (green) and mCherry (red) fluoresce, emitting a yellow fluorescence.

Software:

Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes
Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later. .. Single-plane images were acquired and interactions between mitochondria and ER were calculated using MetaMorph Imaging Series 7.1 image analysis software (Molecular Devices) determining the colocalization index using Mander's coefficient ( ).

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    Addgene inc α synuclein
    Aggregation kinetics of <t>α-synuclein</t> in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.
    α Synuclein, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α synuclein/product/Addgene inc
    Average 93 stars, based on 4 article reviews
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    α synuclein - by Bioz Stars, 2020-04
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    92
    Addgene inc phm6 α synuclein a53t mutant
    Effect of overexpression of α <t>-synuclein</t> on V5-TH1-S31E in neuroblastoma neurites. A , immunofluorescence of neuroblastoma cells co-transfected with V5-TH1-S31E ( red ) and one of the plasmids GFP, His-α-syn WT, or <t>His-α-syn-A53T</t> ( green ) and membranes stained with WGA ( cyan ). B , quantification of the signal of neurites from cells co-transfected with V5-TH1-S31E/GFP V5-TH1-S31E/His-α-syn-WT or V5-TH1-S31E/His-α-syn-A53T. Data are shown as average ± S.D. ( error bars ) and are expressed in arbitrary units ( AU ) ( left ). Representative plot profiles are shown ( right ). For all confocal images, 10-μm scale bars are shown. *, p
    Phm6 α Synuclein A53t Mutant, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phm6 α synuclein a53t mutant/product/Addgene inc
    Average 92 stars, based on 1 article reviews
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    phm6 α synuclein a53t mutant - by Bioz Stars, 2020-04
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    Image Search Results


    Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.

    Journal: Bio-protocol

    Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

    doi: 10.21769/BioProtoc.2941

    Figure Lengend Snippet: Aggregation kinetics of α-synuclein in presence of an inhibitor. 25 μM α-synuclein aggregated with (blue) and without (grey) 25 μM dityrosine (DiY)-crosslinked α-synuclein and 10 μM Thioflavin T and 0.05% sodium azide in SEC buffer, measured in triplicates. A. ThT fluorescence readings of triplicate measurements with and without inhibitor. B. Size exclusion chromatography of samples taken before and after the aggregation assay with and without inhibitor. Note that in presence of the inhibitor, the monomer peak after aggregation is equally high, whereas it decreased markedly without the inhibitor.

    Article Snippet: 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry.

    Techniques: Size-exclusion Chromatography, Fluorescence

    Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).

    Journal: Bio-protocol

    Article Title: α-Synuclein Aggregation Monitored by Thioflavin T Fluorescence Assay

    doi: 10.21769/BioProtoc.2941

    Figure Lengend Snippet: Aggregation kinetics of α-synuclein studied by ThT fluorescence. ). A. Raw data, absolute ThT fluorescence readings of triplicate measurements of the four concentrations; B. Normalized aggregation data of the four concentrations; C. Double logarithmic half-time plot (log( m 0 ): initial monomer concentration vs log(⊺): aggregation half-time).

    Article Snippet: 50 ml Falcon centrifuge tubes (TPP Techno Plastic Products, catalog number: 91050) Sterile syringe filter, Filtropur S, 0.2 μm (SARSTEDT, catalog number: 83.1826.001) Slide-A-Lyzer™ dialysis cassettes 10 kDA MW cutoff, 3-12 ml (Thermo Fisher Scientific, catalog number: 66810) HiTrap Q HP IEC column, 5 ml (GE Healthcare, catalog number: 17115401) Superdex 75 16/60 SEC column, ~120 ml volume (GE Healthcare, catalog number: 28989333) Protein LoBind Tubes 1.5 ml (Eppendorf, catalog number: 0030108116) 96-well plates (Corning half-area, black and clear flat bottom, non-binding surface) (Corning, catalog number: 3881) Sealing tape, clear polyolefin (Thermo Fisher Scientific, catalog number: 232701) E. coli BL21DE3 competent cells ( e.g., available from Merck, catalog number: 69450) α-Synuclein in pT7-7 vector ( e.g., Addgene, catalog number: 36046) We use a version of this plasmid that is codon-optimized for E. coli NatB in pACYCduet vector ( e.g., Addgene, catalog number: 53613) Isopropyl β-D-1-thiogalactopyranoside (IPTG), > 99% (Ubiquitin-Proteasome Biotechnologies, catalog number: P1010-100) Ampicillin sodium salt (Sigma-Aldrich, catalog number: A9518) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) YT medium (2x) premixed powder (AppliChem, catalog number: A0981) Magnesium sulfate heptahydrate p.a. (Merck, catalog number: 1058860500) Glycerol bidistilled, ≥ 99.5% (VWR, catalog number: 24388) Tris(hydroxymethyl)aminomethane, Trizma base p.a. (Sigma-Aldrich, catalog number: 93350) Sodium chloride, p.a. (Carl Roth, catalog number: 3957) Sodium dihydrogen phosphate monohydrate, p.a. (AppliChem, catalog number: 131965.1211) α-Synuclein, N-terminally acetylated (from recombinant expression in E. coli [ ] and purified as described [ ]) Note: We verify N-terminal acetylation by HPLC and mass spectrometry.

    Techniques: Fluorescence, Concentration Assay

    Subcellular localization of α-synuclein. A , In vitro mitochondrial import assays. Import of [ 35 S]Met/Cys-labeled α-syn and a control protein (Mdh2) into HeLa cell mitochondria for the indicated times. p, Precursor protein; m, mature processed protein. Imported proteins were separated by SDS-PAGE. Note that depletion of Δψ affected only the import of Mdh2. B , Western blot to detect the indicated proteins in standard subcellular fractionation of M17 and HeLa cells expressing WT α-syn and in normal human and mouse brain. The CM fraction was purified further into MAM and mitochondrial fractions. ATPase-α (complex V) was used as a mitochondrial marker, protein kinase C as a cytoplasmic marker, and ERLIN-2 as a MAM marker.

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Subcellular localization of α-synuclein. A , In vitro mitochondrial import assays. Import of [ 35 S]Met/Cys-labeled α-syn and a control protein (Mdh2) into HeLa cell mitochondria for the indicated times. p, Precursor protein; m, mature processed protein. Imported proteins were separated by SDS-PAGE. Note that depletion of Δψ affected only the import of Mdh2. B , Western blot to detect the indicated proteins in standard subcellular fractionation of M17 and HeLa cells expressing WT α-syn and in normal human and mouse brain. The CM fraction was purified further into MAM and mitochondrial fractions. ATPase-α (complex V) was used as a mitochondrial marker, protein kinase C as a cytoplasmic marker, and ERLIN-2 as a MAM marker.

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: In Vitro, Labeling, SDS Page, Western Blot, Fractionation, Expressing, Purification, Marker

    Western blot to detect OPA1 in CM fractions from M17 cells stably expressing the indicated forms of α-syn. Mitochondrial ATPase-α was used as a loading control.

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Western blot to detect OPA1 in CM fractions from M17 cells stably expressing the indicated forms of α-syn. Mitochondrial ATPase-α was used as a loading control.

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Western Blot, Stable Transfection, Expressing

    Apposition of ER and mitochondria in HeLa cells. A , Examples of colocalization of ER labeled with GFP-Sec61-β (green), and mitochondria labeled with pDsRed2-mito (red), in HeLa cells transiently expressing WT- and A30P-α-syn. B , Quantitation of colocalization (as in A ) by ImageJ analysis, normalized to the baseline level of apposition value in EV cells (baseline = 100; average of 4 experiments ±SD). Asterisk denotes significance versus EV.

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Apposition of ER and mitochondria in HeLa cells. A , Examples of colocalization of ER labeled with GFP-Sec61-β (green), and mitochondria labeled with pDsRed2-mito (red), in HeLa cells transiently expressing WT- and A30P-α-syn. B , Quantitation of colocalization (as in A ) by ImageJ analysis, normalized to the baseline level of apposition value in EV cells (baseline = 100; average of 4 experiments ±SD). Asterisk denotes significance versus EV.

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Labeling, Expressing, Quantitation Assay

    Expression of α-syn in M17 and HeLa cells. A , Left, Example of a Western blot of the expression of α-syn in EV and stably transfected WT, A53T, and A30P α-syn-expressing M17 cells relative to that of control β-actin. Right, Quantitation of the total α-syn in the blots exemplified at left, in arbitrary units; n = 3; asterisk denotes significance versus WT. B , Expression and quantitation of α-syn in HeLa cells, as in ( A ). In both cases, note reduced expression in A53T cells.

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Expression of α-syn in M17 and HeLa cells. A , Left, Example of a Western blot of the expression of α-syn in EV and stably transfected WT, A53T, and A30P α-syn-expressing M17 cells relative to that of control β-actin. Right, Quantitation of the total α-syn in the blots exemplified at left, in arbitrary units; n = 3; asterisk denotes significance versus WT. B , Expression and quantitation of α-syn in HeLa cells, as in ( A ). In both cases, note reduced expression in A53T cells.

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Quantitation Assay

    Localization of α-syn in M17 cells and transgenic mice. A , B , Western blots to detect the indicated proteins in standard subcellular fractionation of M17 cells stably expressing the indicated α-syn proteins ( A ; quantitation of the ratio of α-syn in the CM:Cyto fractions at right) and in transgenic mice expressing the indicated α-syn species ( B ). C , Localization of α-syn species in DRM and non-DRM fractions. CM (containing mitochondria and MAM) from transgenic mouse brain expressing WT and mutant α-syn were treated with Triton X-100 and then loaded onto a continuous 5–35% sucrose gradient, and the indicated fractions were collected and subjected to Western blotting to detect and quantitate α-syn (see Materials and Methods). Note that α-syn from WT and A53T, but not A30P, mice comigrate with flotillin, a marker of DRMs/lipid rafts.

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Localization of α-syn in M17 cells and transgenic mice. A , B , Western blots to detect the indicated proteins in standard subcellular fractionation of M17 cells stably expressing the indicated α-syn proteins ( A ; quantitation of the ratio of α-syn in the CM:Cyto fractions at right) and in transgenic mice expressing the indicated α-syn species ( B ). C , Localization of α-syn species in DRM and non-DRM fractions. CM (containing mitochondria and MAM) from transgenic mouse brain expressing WT and mutant α-syn were treated with Triton X-100 and then loaded onto a continuous 5–35% sucrose gradient, and the indicated fractions were collected and subjected to Western blotting to detect and quantitate α-syn (see Materials and Methods). Note that α-syn from WT and A53T, but not A30P, mice comigrate with flotillin, a marker of DRMs/lipid rafts.

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Transgenic Assay, Mouse Assay, Western Blot, Fractionation, Stable Transfection, Expressing, Quantitation Assay, Mutagenesis, Marker

    Mitochondrial fragmentation in M17 cells. A , Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B , Quantitation of fragmentation of the cells in A , measured as the percentage of all cells that contained predominantly fragmented (darker shading) versus tubular (lighter shading) mitochondria. Numbers within the boxes denote the average length (±SE) of the mitochondria in micrometers; n = number of mitochondria measured. *Significant difference versus EV ( p

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Mitochondrial fragmentation in M17 cells. A , Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B , Quantitation of fragmentation of the cells in A , measured as the percentage of all cells that contained predominantly fragmented (darker shading) versus tubular (lighter shading) mitochondria. Numbers within the boxes denote the average length (±SE) of the mitochondria in micrometers; n = number of mitochondria measured. *Significant difference versus EV ( p

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Expressing, Labeling, Quantitation Assay

    Mitochondrial fragmentation in HeLa cells. A , Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B , Quantitation of fragmentation of the cells in A , measured as the percentage of all cells that contained predominantly fragmented tubular mitochondria. *Significant difference versus EV ( p

    Journal: The Journal of Neuroscience

    Article Title: ?-Synuclein Is Localized to Mitochondria-Associated ER Membranes

    doi: 10.1523/JNEUROSCI.2507-13.2014

    Figure Lengend Snippet: Mitochondrial fragmentation in HeLa cells. A , Representative images of mitochondria in α-syn-expressing cells labeled with pDsRed2-Mito. Insets, Enlargements of the indicated regions (small boxes). B , Quantitation of fragmentation of the cells in A , measured as the percentage of all cells that contained predominantly fragmented tubular mitochondria. *Significant difference versus EV ( p

    Article Snippet: HeLa cells and Mfn2-WT and -KO MEFs transfected with α-syn-HA constructs, and M17 cells stably expressing α-syn, were cotransfected with GFP-Sec61-β (Addgene; plasmid no. 15108) and DsRed-Mito (Clontech; no. 632421) and fixed 24 h later.

    Techniques: Expressing, Labeling, Quantitation Assay

    Effect of overexpression of α -synuclein on V5-TH1-S31E in neuroblastoma neurites. A , immunofluorescence of neuroblastoma cells co-transfected with V5-TH1-S31E ( red ) and one of the plasmids GFP, His-α-syn WT, or His-α-syn-A53T ( green ) and membranes stained with WGA ( cyan ). B , quantification of the signal of neurites from cells co-transfected with V5-TH1-S31E/GFP V5-TH1-S31E/His-α-syn-WT or V5-TH1-S31E/His-α-syn-A53T. Data are shown as average ± S.D. ( error bars ) and are expressed in arbitrary units ( AU ) ( left ). Representative plot profiles are shown ( right ). For all confocal images, 10-μm scale bars are shown. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation at serine 31 targets tyrosine hydroxylase to vesicles for transport along microtubules

    doi: 10.1074/jbc.M116.762344

    Figure Lengend Snippet: Effect of overexpression of α -synuclein on V5-TH1-S31E in neuroblastoma neurites. A , immunofluorescence of neuroblastoma cells co-transfected with V5-TH1-S31E ( red ) and one of the plasmids GFP, His-α-syn WT, or His-α-syn-A53T ( green ) and membranes stained with WGA ( cyan ). B , quantification of the signal of neurites from cells co-transfected with V5-TH1-S31E/GFP V5-TH1-S31E/His-α-syn-WT or V5-TH1-S31E/His-α-syn-A53T. Data are shown as average ± S.D. ( error bars ) and are expressed in arbitrary units ( AU ) ( left ). Representative plot profiles are shown ( right ). For all confocal images, 10-μm scale bars are shown. *, p

    Article Snippet: Soluble enhanced green fluorescent protein (GFP) was purchased from Clontech. pHM6-α-synuclein-A53T mutant was a gift from David Rubinsztein (Addgene plasmids 40824 and 40825) and described previously ( ).

    Techniques: Over Expression, Immunofluorescence, Transfection, Staining, Whole Genome Amplification