Chinese Bulletin of Botany ›› 2021, Vol. 56 ›› Issue (5): 584-593.DOI: 10.11983/CBB21104
• INVITED PROTOCOL • Previous Articles Next Articles
Jiayi Kuang, Hongqing Li, Wenjin Shen*(), Caiji Gao*()
Received:
2021-07-01
Accepted:
2021-08-09
Online:
2021-09-01
Published:
2021-08-31
Contact:
Wenjin Shen,Caiji Gao
Jiayi Kuang, Hongqing Li, Wenjin Shen, Caiji Gao. Methods for TurboID-based Proximal Labeling in Plants[J]. Chinese Bulletin of Botany, 2021, 56(5): 584-593.
Solution | Composition | Working concentration |
---|---|---|
Protein Extraction Buffer | Tris-HCl (pH7.5) | 50 mmol∙L-1 |
NaCl | 150 mmol∙L-1 | |
Sodium deoxycholate | 0.5% (w/v) | |
SDS | 0.1% (w/v) | |
EDTA | 1 mmol∙L-1 | |
Triton X-100 | 1% (v/v) | |
DTT | 1 mmol∙L-1 | |
PMSF | 1 mmol∙L-1 | |
Leupeptin | 10 μg∙mL-1 | |
Protease Inhibitor Cocktail | 1× | |
4×SDS Sample Buffer | Tris-HCl (pH6.8) | 200 mmol∙L-1 |
SDS | 8% (w/v) | |
Glycerol | 40% (v/v) | |
β-mercaptoethanol | 20% (v/v) | |
Bromophenol blue | 0.1% (w/v) | |
1×PBS buffer (pH7.4) | NaH2PO4∙2H2O | 0.263 g∙L-1 |
Na2HPO4∙12H2O | 1.856 g∙L-1 | |
NaCl | 8 g∙L-1 | |
KCl | 0.2 g∙L-1 | |
1×PBST | 1×PBS buffer containing 0.05% Tween-20 | |
Biotin stock (stored at -20°C) | Dissolve biotin in DMSO to a final concentration of 50 mmol∙L-1 |
Table 1 Related reagent formulations used in experiment
Solution | Composition | Working concentration |
---|---|---|
Protein Extraction Buffer | Tris-HCl (pH7.5) | 50 mmol∙L-1 |
NaCl | 150 mmol∙L-1 | |
Sodium deoxycholate | 0.5% (w/v) | |
SDS | 0.1% (w/v) | |
EDTA | 1 mmol∙L-1 | |
Triton X-100 | 1% (v/v) | |
DTT | 1 mmol∙L-1 | |
PMSF | 1 mmol∙L-1 | |
Leupeptin | 10 μg∙mL-1 | |
Protease Inhibitor Cocktail | 1× | |
4×SDS Sample Buffer | Tris-HCl (pH6.8) | 200 mmol∙L-1 |
SDS | 8% (w/v) | |
Glycerol | 40% (v/v) | |
β-mercaptoethanol | 20% (v/v) | |
Bromophenol blue | 0.1% (w/v) | |
1×PBS buffer (pH7.4) | NaH2PO4∙2H2O | 0.263 g∙L-1 |
Na2HPO4∙12H2O | 1.856 g∙L-1 | |
NaCl | 8 g∙L-1 | |
KCl | 0.2 g∙L-1 | |
1×PBST | 1×PBS buffer containing 0.05% Tween-20 | |
Biotin stock (stored at -20°C) | Dissolve biotin in DMSO to a final concentration of 50 mmol∙L-1 |
Figure 2 Subcellular localization of TurboID-EYFP-ATG8a in stable Arabidopsis lines 6-day-old transgenic Arabidopsis seedlings overexpressing TurboID-EYFP-ATG8a were transferred to nitrogen (N)- deficient medium with 1 µmol∙L-1 Conc A for 8 h followed by confocal microscopic analysis. Bars=50 μm
Figure 3 Immunoblot analysis of biotinylated protein (A) Wild-type (WT), transgenic Arabidopsis seedlings overexpressing TurboID-EYFP (TY/WT) or TurboID-EYFP-ATG8a (TY-8A/WT) were treated with 50 µmol∙L-1 biotin (+) for 3 h or 0 (-), activity and expression of TurboID-EYFP construct were analyzed by immunoblots with SA-HRP and anti-GFP antibodies; (B) Different lines of transgenic Arabidopsis seedlings overexpressing TurboID-EYFP (TY/WT) were treated with 50 µmol∙L-1 biotin (+) for 3 h or 0 (-), followed by immunoblots. SA-HRP: Streptavidin-HRP; CBB: Coomassie Brilliant Blue
Figure 4 The optimization of biotin concentration and labeling time used in proximity labeling (A) Wild-type (WT) and transgenic Arabidopsis seedlings overexpressing TurboID-EYFP were treated with different concentrations of biotin solution for 1 h, followed by immunoblots with SA-HRP and anti-GFP antibodies to detect the proximity ligation activity and protein expression level of TurbolD-EYFP; (B) Wild-type (WT) and transgenic Arabidopsis seedings overexpressing TurboID-EYFP were treated with 50 µmol∙L-1 biotin for different times, followed by immunoblots with appropriate antibodies. SA-HRP: Streptavidin-HRP; CBB: Coomassie Brilliant Blue
Figure 5 Immunoblotting analyses of the biotinylated proteins enriched by Streptavidin Magnetic Beads Arabidopsis seedlings overexpressing TurboID-EYFP-ATG8a were treated with 50 µmol∙L-1 biotin for 3 hours. The protein was extracted and desalted through Desalting Column. The desalted sample was split into two parts and incubated with 20 μL Streptavidin Magnetic Beads overnight, followed by protein elution with 20 μL or 200 μL 4× SDS Sample Buffer, respectively. Extract: Protein supernatant after centrifugation; After Desalting Column: Protein sample after desalting; Flow-through 1 or 2: Supernatant after overnight incubation with streptavidin (SA) beads; Eluate 1: Protein sample eluated with 20 μL buffer; Eluate 2: Protein sample eluated with 200 μL buffer; SA-HRP: Streptavidin-HRP
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