All experiments were performed as described previously (Kolonko et al., 2020 (link)). Briefly, samples obtained during the pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich). The membranes were blocked with milk powder (Milchpulver, blotting grade, Roth), then incubated overnight with specific primary anti-GFP polyclonal antibodies (1:5,000, cat. no. SAB2702197, Sigma-Aldrich) or anti-14-3-3 antibodies (1:5,000, cat. no. AB9748-I, Millipore), followed by the incubation with secondary horse anti-mouse antibodies (for anti-GFP, 1:10,000, cat. no. PI-2000, Vector Laboratories) or goat (for anti-14-3-3, 1:10,000, Sigma-Aldrich, cat. no. A 6154) anti-mouse antibodies coupled to horseradish peroxidase. Signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescent kit (Thermo Scientific™). Finally, the membranes were exposed to Kodak BioLight film. The MM of each fusion protein was calculated based on aa sequences using the ProtParam server (Gasteiger, 2019 ) (YFP-Met-FLAG 107 kDa, YFP-Gce-FLAG 105 kDa, YFP-MetC-FLAG 51.4 kDa, YFP-GceC-FLAG 64 kDa, CFP-Ftz-F1 LBD 55 kDa, and CFP-14-3-3 57 kDa).
Milchpulver
Manufactured by Carl Roth
Milchpulver is a powdered form of milk produced by Carl Roth. It is a dehydrated dairy product that is created through the removal of water from fresh liquid milk.
Automatically generated - may contain errors
Lab products found in correlation
Sab2702197, by Merck Group (1 mentions)
Specific primary anti gfp polyclonal antibodies, by Merck Group (1 mentions)
Goat anti mouse antibodies coupled to horseradish peroxidase, by Vector Laboratories (1 mentions)
Supersignal west pico plus chemiluminescence substrate kit, by Thermo Fisher Scientific (1 mentions)
3 protocols using milchpulver
1
Western Blot Analysis of Protein Interactions
2
Western Blot Analysis of Fluorescent Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature.
Next, the membrane was incubated overnight at 4 °C with the speci c primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3x10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10 000 with milk buffer). Speci c signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scienti c™) according to the manufacturer's manual. Finally, the membranes were exposed to Kodak BioLight lm.
Next, the membrane was incubated overnight at 4 °C with the speci c primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3x10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10 000 with milk buffer). Speci c signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scienti c™) according to the manufacturer's manual. Finally, the membranes were exposed to Kodak BioLight lm.
3
Western Blotting of Fluorescent Fusion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the samples obtained during the FLAG Pull-down assay were separated by SDS-PAGE using 12% gels and transferred to the Whatman Protran nitrocellulose transfer membrane (Protran BA85, Schleicher & Schuell Pure, Sigma-Aldrich) in the semi-dry system at 10 V for 40 min in Towbin buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). The membranes were blocked at room temperature with 2% milk powder (Milchpulver, blotting grade, Roth) in the PBS buffer and incubated for 1 h at room temperature. Next, the membrane was incubated overnight at 4 °C with the specific primary anti-GFP polyclonal antibodies (Sigma-Aldrich) (diluted 1:300 with milk buffer), which cross-react with CFP and YFP. After washing (PBS supplemented with 0.02% Tween, 3x10 min), the membrane was incubated for 2 h with secondary goat anti-mouse antibodies coupled to horseradish peroxidase (Vector Laboratories, dilution 1:10 000 with milk buffer). Specific signals were detected using the SuperSignal™ West Pico PLUS Substrate Chemiluminescence kit (Thermo Scientific™) according to the manufacturer's manual. Finally, the membranes were exposed to Kodak BioLight film.
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