HEK293 cells were transfected in 6-well plates and cultured for 48 h. Cells were lysed for 10 min at 4 °C with 100 mM Tris pH 7.5, 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, and protease inhibitor cocktail. Cell lysates were cleared by centrifugation at 10,000×g for 3 min at 4 °C. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo Blotting apparatus (Bio-Rad). Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-GFP (Clontech cat. no. 632380, 1:8000, for YFP constructs); anti-Myc tag (Abcam cat. no. ab9106, 1:1000); and anti-β-actin (Sigma cat. no. A5441, 1:10,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and a ChemiDoc XRS+ imaging system (Bio-Rad).
Transblot turbo blotting apparatus
Manufactured by Bio-Rad
The TransBlot Turbo Blotting apparatus is a compact and efficient device designed for the transfer of proteins from polyacrylamide gels to membranes. It utilizes a rapid and consistent blotting process to facilitate the analysis of proteins in research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp, by Takara Bio (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Chemidoc xrs imaging system, by Bio-Rad (3 mentions)
Novex ecl chemiluminescent substrate reagent kit, by Thermo Fisher Scientific (3 mentions)
Anti myc tag, by Abcam (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Ponceau s staining, by Merck Group (1 mentions)
Anti mcherry, by Novus Biologicals (1 mentions)
Chemidoc xrs system image analysis software, by Bio-Rad (1 mentions)
5 protocols using transblot turbo blotting apparatus
1
Western Blot Analysis of Transfected HEK293 Cells
2
Western Blotting and Immunocytochemistry Protocol
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For western blotting analysis, conventional SDS-PAGE and immunoblotting techniques were used. Briefly protein lysates were prepared in RIPA lysis buffer containing: 50 mM Tris 1% NP40, 1% SDS, 0.25% sodium deoxycholate and protease and phosphatase inhibitor cocktails (Sigma). SDS-PAGE separation was performed using the Life Technologies Novex NuPAGE SDS-PAGE gel system and transfer was performed using the Bio-Rad Trans-Blot Turbo blotting apparatus.
For immunocytochemistry, neurons were fixed in 4% PFA, washed three times and incubated with primary antibody for 4 h at room temperature. SMI-312 antibody (1:1000) was applied in 0.3% Triton X-100, 5% BSA, PBS solution after an additional permeabilization step with 0.5% NP40/PBS solution for 15 min. After washing samples three times with 0.1% Triton X-100/PBS solution, secondary Alexa Fluor conjugated antibody (1:500) was applied for 30 min. Stained cells were stabilized using 80% glycerol solution.
For immunocytochemistry, neurons were fixed in 4% PFA, washed three times and incubated with primary antibody for 4 h at room temperature. SMI-312 antibody (1:1000) was applied in 0.3% Triton X-100, 5% BSA, PBS solution after an additional permeabilization step with 0.5% NP40/PBS solution for 15 min. After washing samples three times with 0.1% Triton X-100/PBS solution, secondary Alexa Fluor conjugated antibody (1:500) was applied for 30 min. Stained cells were stabilized using 80% glycerol solution.
3
Western Blot Analysis of Exogenous Proteins
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Cells were transfected in 6-well plates and cultured for 48 hr. Cells were lysed for 10 min at 4°C with 100 mM Tris (pH 7.5), 150 mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1% PMSF, and protease inhibitor cocktail. Cell lysates were cleared by centrifugation at 10,000 × g for 3 min at 4°C. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo blotting apparatus (Bio-Rad). Membranes were blocked in phosphate-buffered saline containing 5% non-fat milk powder and 0.1% Tween-20 and then incubated overnight at 4°C with primary antibody. The following antibodies were used: anti-GFP (Clontech cat# 632380, RRID: AB_10013427 ; 1:8,000, for YFP constructs) and anti-β-actin (Sigma cat# A5441, RRID: AB_47644 ; 1:10,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen) and a ChemiDoc XRS+ imaging system (Bio-Rad).
4
Western Blot Protein Detection Protocol
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Cells were lysed with RIPA buffer supplemented with 5 mM sodium butyrate and 1× Protease Inhibitor Cocktail (Thermo Scientific). Protein extracts were resolved using SDS-PAGE and transferred to nitrocellulose membranes, using the Trans-Blot Turbo blotting apparatus and reagents (all from Bio-Rad). Protein transfer was confirmed by Ponceau S staining (Sigma-Aldrich). The membranes were then blocked using 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween20 (TBS-T) for 1 h at RT and were probed with the indicated primary antibodies, diluted in 3% milk in TBS-T at 4 °C overnight. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies diluted in 5% BSA in TBS-T, for 1 h at RT. Detection was performed providing fresh horseradish peroxidase-substrate solution (Luminol Enhancer Solution/Peroxide Solution; Promega) and exposing of membranes for specific time periods to photographic film using a Curix60 instrument (Agfa).
5
Western Blot Analysis of Protein Expression
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HEK293 cells were transfected in 6-well plates and cultured for 48 h. Cells were lysed in 300 μL of Laemmli sample buffer containing 10% Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and incubated for 10 min at 95 °C. Proteins were resolved on 10% SDS-polyacrylamide gels and transferred to PVDF membranes using a TransBlot Turbo Blotting apparatus (Bio-Rad). Membranes were blocked in PBS containing 5% milk and 0.1% Tween-20 and incubated overnight at 4 °C with primary antibody. The following antibodies were used: anti-GFP (Clontech cat. no. 632380, 1:8000, for YFP constructs); anti-mCherry (Novus cat. no. NBP1-96751, 1:1000); anti-V5 tag (Genetex cat. no. GTX42525, 1:3000); anti-Myc tag (Abcam cat. no. ab9106, 1:1000); anti-β-actin (Sigma cat. no. A5441, 1:10,000). After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG for 45 min at room temperature. Proteins were visualized using Novex ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) and a ChemiDoc XRS + imaging system (Bio-Rad). Densitometry was performed using the Chemidoc XRS + System image analysis software (Bio-Rad).
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