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Team:HKUST/Protocols/Western blotting
From 2009.igem.org
Western Blotting
- 1. Yeast transformed cells containing appropriate plasmids were grown in SCM with appropriate amino acids deleted and harvested at a cell density corresponding to an OD600 of a 10-fold dilution reaching 0.2.
- 2. 2× Laemmli’s buffer (20% v/v glycerol, 0.1M Tris-HCl pH 6.9, 4% SDS, 0.2% bromophenol blue, 10% mercaptoethanol) was added and yeast crude extracts were prepared by glass bead lysis and separated on 10% SDS-PAGE.
- 3. The 10% resolving gel and the 5% stacking gel were cast according to the method described in Molecular Cloning (Sambrook et al., 1989).Separation was performed on a Mini Protean 3 gel and transblot system (Biorad). Proteins were stacked at 60-80 volts in the stacking gel and resolved at 120-150 volts in the resolving gel.
- 4. The proteins were then transferred onto a nitrocellulose membrane (pore size 0.45 μm, Schleicher &Schuell) in electrophoretic blotting system (C.B.S. Scientific) by applying a constant current of 0.5 mA for 1.5 hours.
- 5. After transferring, the membrane was taken out and socked in Ponceau S solution (0.5% Ponceau S in 1% HAc) for 1 min. After rinsed with ddH2O and labeled with protein marker bands.
- 6. The membrane was destained with TBST buffer (10 mM Tris-HCl pH8.0, 150 mM NaCl, 0.05% v/v Tween-20) and blocked with 5% milk in TBST for 30 min.
- 7. Then the membrane was incubated with the primary antibody (mouse monoclonal antibody against Flag) for 1 hour at room temperature followed by 3 × 15 min washing with TBST.
- 8. The membrane was then incubated with secondary antibody (goat anti-mouse antibody conjugated with horseradish peroxidase) for 1 hour at room temperature followed by 3 × 15 min washing with TBST.
- 9. After that, the membrane was incubated in SuperSignal chemiluminescence substrate (Pierce) for 5 min and exposed to light-sensitive films (Fuji). In the end, the film was developed in a film-processing machine (Eastman Kodak).
