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More western blot protocols

Immunoblot detection
(Curr Protoc Protein Sci. 2001 May;Chapter 10:Unit 10.10)
Gallagher S.

Hoefer Scientific Instruments, San Francisco, California, USA.

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.

Identification of protein interactions by far western analysis
(Curr Protoc Protein Sci. 2001 Nov;Chapter 19:Unit 19.7)
Edmondson DG, Dent SY.

MD Anderson Cancer Center, Houston, Texas, USA.

This unit describes far western blotting, a method of identifying protein-protein interactions. In a far western blot, one protein of interest is immobilized on a solid support membrane, then probed with a non-antibody protein. As described, far western blots can be used to identify specific interacting proteins in a complex mixture of proteins. They are particularly useful for examining interactions between proteins that are difficult to analyze by other methods due to solubility problems or because they are difficult to express in cells. This method is performed totally in vitro, and the proteins of interest can be prepared in a variety of ways. A protocol is also provided for determining the effects of specific peptide residues or post-translational modifications on protein-protein interactions. Many different detection techniques, either radioactive or nonradioactive, can be used. For example, the protein probe may be detected indirectly with an antibody, rather than being labeled radioactively

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels
(Journal of visualized experiments. Aubin Penna, Michael Cahalan Department of Physiology and Biophysics, University of California, Irvine)
Excellent video protocol for western blotting using NuPage minigel system.

Western Blotting with Monoclonal Antibodies
(BD Biosciences technotes.)
Western blotting protocol including sample preparation, blotting and troubleshooting using monoclonal antibodies.

SDS-PAGE electrophoresis and western blotting
(UC Davis by Vinh Pham.)
Western blotting protocol.

Tris and transfer buffer
FAQ on western blotting transfer membrane and transfer buffer system

western blot stripping buffer
Stripping buffers use either pH, heat or detergent to remove antibody

Wet transfer vs Semi-dry transfer
Comparison of wet transfer and semi-dry transfer

Problems with stripping a western blot
reprobing reveals only faint traces when overexposed

Western blot with rabbit polyclonal antibodies
used purified E. coli protein to generate polyclonal antibodies from rabbit

Quantifying Western Blots
CCD camera set-up to obtain western blot images and then processed and quantified the data (IDV) using Scion Image

speckled background on westerns
random specks andtiny smears all over the membrane

Western blot with HA-tagged protein expression
I tried to express some HA-tagged proteins in K562 cells many times, 4 different proteins actually...

Western blot using recombinant protein
Multiple bands on western blot

Western blotting
Actin antibody

GAPDH normalization for western blotting
internal standards normalization

hard-to-detect proteins in mammalian cells
Western blot detection of low abundant proteins

No band at film developing ( Western blot)
No signal troubleshooting

high background and white bands in western blot
Western blotting high background

triton is affecting protein detection
Triton x-100 in washing buffer

western blot problems
Western blot troubleshooting

Western blot funneling
rat white fat samples western blot

Storage options for nitrocellulose membranes
nitorcellulose membrane for western blot

Glycoprotein and Western Blot
Recombinant Glycoprotein Western Blot

membrane protein extraction for western blot
Western blot and membrane protein extraction

Two bands in western blot
Western blotting non-specific bands

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