Home Technique / Proteomics and protein biochemistry / Western blot assay

Western blot protocol new protocol
Preparation of cell lysates from E.Coli bacteria for western blotting: 1. Spin down 1 ML overnight culture 2. add 100-200 ul BugBuster protein extraction reagent (Novagen cat. No. 70584) 3. Sonicate 1 min 4. add 2x sample buffer to same lysis volume(or a ...

Multiplex western blot using quantum dot fluorescence technology new protocol
A technique note on multiplex western blot analysis using Qdot fluorescent technology. Nature Methods 2, 79 - 81 (2005) Abstract: Western or immunoblotting analysis of protein expression in cells and tissues has been the major analytical tool for as ...

Western blot stripping protocol new protocol
Western blot stripping protocol: Buffer needed: stripping buffer: 2% SDS 100 mM beta-mercaptoethanol 50 mM Tris, pH 6.8 Procedure: 1. Heat stripping buffer to 50c in water bath. 2. Incubate blot with stripping buffer at 50c for 15-30 min with gen ...

Semi-dry western blot transfer procedure new protocol
Semi-dry western blot transfer procedure: This protocol describes the procedure for transfering protein from polyacrylamide gel onto nylone membrane using semi-dry transfer method. 1. Cut 6-10 sheets of whatman 3MM the same size as the mini PAGE gel. ...

Western blot new review
A typical western blot procedure includes following steps: 1. Protein lysate preparation from cells and tissues. 2. Protein assay for determination of protein concentration in lysate. 3. Denaturing and reduction of protein complexes before loading onto ge ...

large protein transfer efficiency for western blot new hot troubleshooting
A newsgroup post on large protein transfer efficiency for western blot. I have been having a lot of problems with efficiency of transfer from gel (10% acrylamide/bis) to PVDF membrane (Immobilon P). When I stain the gel after transfer, there is a lot o ...

Homemade Chemiluminescent Protocols? troubleshooting
Company kits end up costing $3-5 per western blot which is 90% of the cost of the whole procedure. People must have some good homemade HRP (and AlkPhos) Chemiluminescent Detection protocols or references? ... ... Click follow ...

Western-Blot: PBS/TBS troubleshooting
Does anybody know if it makes a difference for certain antibodys (for protein-detection on a western-blot) to use PBS instead of TBS as basic for all the incubation- and washing-buffers? The reason why I´m asking is the following one: ... ...

Weird Western Blot (circular streaks) troubleshooting
I've had a couple of weird western blots lately, we use a standard SDS PAGE tris glycine minigel (BIORAD), and transfer using the minigel apparatus at 100v for 90 minutes with a cooling block (ice in a container) present. The transfer buffer is ...

Western blot problem hot troubleshooting
I have been having a lot of problems with efficiency of transfer from gel (10% acrylamide/bis) to PVDF membrane (Immobilon P). When I stain the gel after transfer, there is a lot of protein left on there. I know that I am not exceeding the binding cap ...

Troubleshooting the Western blot procedure troubleshooting
From Roche Applied Biosciences (PDF file), ...

Troubleshooting Guide: Western Blot new troubleshooting
From R&D systems. It gives troubleshooting guide on several common problems on western blot: low protein transfer, high background, low protein binding, multiple bands staining etc. ...

Multiple bands on a Western blot and solutions for western blot background new troubleshooting
A FAQ from Synaptic System on problem of many bands on a western blot. Beside solutions provided above, some other considerations may be helpful for eliminate multiple bands in western blot. Some solutions for high background of western blot (multiple ba ...

Western Blot troubleshooting new troubleshooting
Western blot troubleshooting tips - protein transfer problem: poor transfer efficiency, air bubbles trapped when assembling the transfer sandwitch (use a tube or roller to get ride of any trapped air bubbles), voltage and current is not optimized for ...

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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