Team:HKUST/Protocols/Western blotting

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Lab Notebook</a></li> Parts Submitted </a></li> Protocol List</a></li> Other Resources</a></li> </ul> <ul> <li><a href="http://2009.igem.org/Team:Gallery">Gallery</a></li> <li><a href="http://2009.igem.org/Team:Biosafety">Biosafety</a></li> <li><a href="http://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li> </ul> a

Western Blotting

Procedure: 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).

<ul> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel electrophoresis</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to yeast</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA extraction</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li> <li><a href="http://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li>

</ul> iGEM 2009

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