Chinese Bulletin of Botany ›› 2020, Vol. 55 ›› Issue (1): 62-68.DOI: 10.11983/CBB19143
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Received:
2019-08-01
Accepted:
2019-08-30
Online:
2020-01-01
Published:
2019-12-20
Contact:
Chongyi Xu
Chongyi Xu. Pull-down and Co-immunoprecipitation Assays of Interacting Proteins in Plants[J]. Chinese Bulletin of Botany, 2020, 55(1): 62-68.
Solution | Composition | Final concentration |
---|---|---|
1× PBS buffer (pH7.4) | NaCl KCl | 137 mmol·L-1 2.7 mmol·L-1 |
Na2HPO4 | 10 mmol·L-1 | |
KH2PO4 | 2 mmol·L-1 | |
NETN buffer | Tris-HCl (pH8.0) | 20 mmol·L-1 |
NaCl | 100 mmol·L-1 | |
EDTA | 0.5 mmol·L-1 | |
Nonidet P-40 (NP-40) | 0.5% (v/v) | |
3× SDS sam- ple buffer | Tris-HCl (pH6.8) Glycerin | 167 mmol·L-1 33% (v/v) |
Sodium dodecyl sulfate | 6.6% (w/v) | |
Bromophenol blue | 0.01% (w/v) | |
β-mercaptoethanol | 7.5% (v/v) |
Table 1 Related reagent formulations used in the GST Pull- down
Solution | Composition | Final concentration |
---|---|---|
1× PBS buffer (pH7.4) | NaCl KCl | 137 mmol·L-1 2.7 mmol·L-1 |
Na2HPO4 | 10 mmol·L-1 | |
KH2PO4 | 2 mmol·L-1 | |
NETN buffer | Tris-HCl (pH8.0) | 20 mmol·L-1 |
NaCl | 100 mmol·L-1 | |
EDTA | 0.5 mmol·L-1 | |
Nonidet P-40 (NP-40) | 0.5% (v/v) | |
3× SDS sam- ple buffer | Tris-HCl (pH6.8) Glycerin | 167 mmol·L-1 33% (v/v) |
Sodium dodecyl sulfate | 6.6% (w/v) | |
Bromophenol blue | 0.01% (w/v) | |
β-mercaptoethanol | 7.5% (v/v) |
Solution | Composition | Final concentration |
---|---|---|
Infiltration buffer | MES-KOH (pH5.6) | 10 mmol·L-1 |
MgCl2 | 10 mmol·L-1 | |
Acetyl syringone (AS) | 0.2 mmol·L-1 | |
NB1 buffer | Tris-MES (pH8.0) | 50 mmol·L-1 |
Sucrose | 500 mmol·L-1 | |
MgCl2 | 1 mmol·L-1 | |
EDTA | 10 mmol·L-1 | |
DTT | 5 mmol·L-1 | |
PMSF | 1 mmol·L-1 | |
Cocktail | 100× | |
3× SDS sample buffer | Same as Pull-down |
Table 2 Related reagent formulations used in the Co-IP
Solution | Composition | Final concentration |
---|---|---|
Infiltration buffer | MES-KOH (pH5.6) | 10 mmol·L-1 |
MgCl2 | 10 mmol·L-1 | |
Acetyl syringone (AS) | 0.2 mmol·L-1 | |
NB1 buffer | Tris-MES (pH8.0) | 50 mmol·L-1 |
Sucrose | 500 mmol·L-1 | |
MgCl2 | 1 mmol·L-1 | |
EDTA | 10 mmol·L-1 | |
DTT | 5 mmol·L-1 | |
PMSF | 1 mmol·L-1 | |
Cocktail | 100× | |
3× SDS sample buffer | Same as Pull-down |
Figure 1 Schematic representation of the Pull-down procedure in prokaryotic expression system to test the interaction between a bait protein tagged with GST (glutathione S-transferase) and a prey protein IPTG: Isopropyl-β-D-thiogalactopyranoside; GSH: Glutathione
Figure 2 Schematic representation of the Co-immunoprecipitation procedure from infiltrated Nicotiana benthamiana leaves to test the interaction between a prey protein tagged with Flag and a bait protein tagged with Myc
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