Overview: Western Blotting or immunoblotting (Western blotting was dubbed because it is similar to Southern blotting which was invented by and named after the inventor E. M. Southern) allows investigators to determine, with a specific primary antibody, the relative amounts of the protein present in different samples. Briefly, 1) Samples are prepared from tissues or cells that are homogenized in a buffer that protects the protein of interest from degradation; 2) The sample is separated using SDS-PAGE and then transferred to a membrane for detection; 3) The membrane is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the membrane. A primary antibody is then added to the solution which is able to bind to its specific protein; 4) A secondary antibody-enzyme conjugate, which recognizes the primary antibody is added to find locations where the primary antibody bound.
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Protocols
- Cheap Chemiluminescent Detection (Mellor Laboratory Bristol)
Using a chemiluminescent reagent mix described by Yakunin and Hallenbeck (1998). The reagents are simple to prepare and cost a fraction of commercial preparations.
http://www.bch.bris.ac.uk/staff/Mellor/docs/protoc...
Added: Sun Jun 05 2005, Hits: 996, Reviews: 0 Write review Cached -
Introduction to Western Blotting (Millipore)
Nice introduction to Western blotting analysis.
http://www.millipore.com/immunodetection/id3/weste...
Added: Wed Feb 13 2008, Hits: 4708, Reviews: 0 Write review Cached - Western Blot Analysis for Tissue (Laboratory of Vet.Biochemistry and Molecular Biology, Chungbuk National University, Republic of Korea)
This protocol was used for tissues in detail to detect signals of protein
Added: Tue May 10 2005, Reviews: 0 Write review - Western Blotting (WB) - A Beginner's Guide
(Abcam)
In this 9-page protocol, you can find everything you need to know about Western blotting including lysate preparation, denaturation and reduction of protein, electrophoresis (tips on positive controls, MW markers, loading controls), tips on transferring, blocking and incubation.
http://www.abcam.com/ps/pdf/protocols/WB-beginner....
Added: Sat Jan 07 2006, Hits: 23940, Reviews: 0 Write review - Western Blotting Protocol (Lab of Dr. Mark Barton Frank, Oklahoma Medical Research Foundation)
The most complete Western protocol including:
Preparation of protein from human cells
Preparation of Bacterially Expressed Proteins
SDS-PAGE Separating Gels (Hoeffer baby gels)
Electrophoretic Transfer for Dry Blots
Electrophoretic Transfer For Wet Blots
Detection of Antigen on Membranes
Photograph the membranes
Recipes
http://omrf.ouhsc.edu/~frank/Western.html
Added: Thu Sep 01 2005, Hits: 27274, Reviews: 0 Write review Cached - Western Blotting Protocols (Millipore)
Western immunoblotting protocol for general use including Standard and Rapid Immunodetection Procedures, Membrane Stripping Protocols, Peptide Inhibition/Competition, blot storage
http://www.millipore.com/immunodetection/id3/weste...
Added: Tue Feb 17 2004, Hits: 10426, Reviews: 0 Write review Cached -
Western Blot
(R&D System)
Provides very nice and general protocol including Western Blot Procedure, immunostaining Procedure (standard and rapid), helpful hints and trouble shooting guide
http://www.rndsystems.com/asp/g_sitebuilder.asp?bo...
Added: Tue May 14 2002, Hits: 32826, Reviews: 0 Write review Cached -
Western Blot
(Bjorkman Group, Howard Hughes Medical Institute at California Institute of Technology)
Describes sample preparation, SDS-PAGE, staining, transfer, blotting
http://www.its.caltech.edu/%7Ebjorker/Western_blot...
Added: Wed Aug 14 2002, Hits: 33170, Reviews: 0 Write review -
Anti-Phosphotyrosine Western Blotting
(Bowtell Lab Manual, Peter MacCallum Cancer Institute)
http://www.pmci.unimelb.edu.au/core_facilities/man...
Added: Tue May 14 2002, Hits: 2166, Reviews: 0 Write review Cached -
Extracts for Yeast Westerns
( )
(Herskowitz Lab
http://www.sacs.ucsf.edu/home/HerskowitzLab/protoc...
Added: Tue May 14 2002, Hits: 1129, Reviews: 0 Write review
Trouble Shootings and FAQs
- Can formalin fixed tissue used for Western blots?
- What difference in the band pattern between protein sample with and withouth boiling?
- How to eliminate albumin from samples for western blotting?
- What is the detection threshold of western blotting?
- How to concentrate protein from culture medium for Western blot
- Semi-dry transfer vs wet transfer- which is better?
- Western Blotting - High background troubleshooting
- Western blotting using alkaline phosphatase
- Western Blotting - problem with an internal control
- Western Blot Transfer Problem - no more markers!- did they 'blow through?"
- Western blot transfer trouble
- Primary antibody incubation time?
- Denaturing protein - protein won't fall apart
- Interpreting non-specific bands on Western blot
- Semi dry transfer - Influence of isoelectric point on transfer
- Polyclonal vs monoclonal antibody for Western and ChIP