The transfer of macromolecules, such as nucleic acids and proteins, to solid-phase membranous support is termed blotting. Proteins resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) are transferred to membrane composed of either nitrocellulose or polyvinylidine diflouride (PVDF) using electric current. Also referred to as protein blotting or immunoblotting, Western blotting is a powerful method for studying proteins in a sample of cells or tissue extract. The method involves five main steps detailed below.
Preparing the sample includes protein extraction from cells or tissues and estimation of protein concentration. Various lysis buffers containing detergents and protease and phosphatase inhibitors may be used to solubilize proteins from whole tissue or tissue culture extracts. The total protein in the samples may be quantified spectrophotometrically at 595 nm using Bradford reagent.
Proteins may be separated based on isoelectric point, molecular weight, electric charge or a combination of all these. The most popular method is electrophoresis using polyacrylamide gels loaded with SDS (SDS-PAGE). The protein samples are denatured by boiling with a reducing agent to break the disulfide bonds before electrophoresis.
The proteins separated by gel electrophoresis are immobilized by transfering to a solid support, such as PVDF or nitrocellulose membrane. The transfer uses electric current to pull proteins from the gel onto the membrane (electroblotting). View the SNAP i.d.® 2.0 Blot Roller and other accessories.
For detection of a target protein transferred onto the membrane, it is important to block the detection of any non-specific proteins that results in false-positive results. This is usually done by using blocking agents such as BSA, non-fat milk or specific antiserum. Thereafter, the membrane is incubated with optimized concentrations of primary and secondary antibodies specific to the target protein.
Proteins of interest may be detected by colorimetric, radioactive, chemiluminescent or fluorescent methods. The secondary antibody is typically conjugated to horseradish peroxidase that cleaves a chemiluminescent agent. The luminescence produced by the reaction product is proportional to the amount of the target protein.
Figure 1:Sigma-Aldrich Blotting and Vertical Electrophoresis System
Figure 2. Steps involved in western blotting procedure
All the steps for protein extraction from cells or tissue (fresh or frozen) must be performed at 2-8 °C. The following is the composition of a common lysis buffer used to prepare protein samples.
The following is the composition of loading buffer required to prepare the samples for electrophoresis.
2X Laemmli loading buffer:
Running buffer that acts as both anode and cathode buffer is available (Product No. GE28-9902-52). Alternatively, running buffer can be prepared from individual components.
To confirm the migration and separation of proteins, the gel may be stained with a reversible stain such as CuCl2.
The transfer of proteins from the gel to a flexible and easily-handled membrane support can be performed rapidly using electricity, a process called as electroblotting. Electroblotting can be performed in wet or semi-dry conditions. In both procedures, the main components required are the transfer buffer and the transfer unit.
Before preparing the transfer setup, the following equilibration steps are required for PVDF membranes:
DO NOT incubate nitrocellulose membranes in methanol. Incubation in transfer buffer is sufficient.
To transfer the proteins separated from the gel to the membrane, the following stack has to be prepared in blotter unit. Ensure no air bubbles are trapped between the gel and the membrane.
Figure 3.Western blot transfer assembly
We offer semi-dry blotter units that conserve buffer volume, produce less heat, less band distortion and allows for the transfer of multiple gels of varying thickness.
The transfer can be performed at a voltage of 50 V at 4 °C for 2 h.
The transfer of proteins from gel to membrane may be confirmed by using a reversible membrane stain such as Ponceau S. The following are the reagents required for reversible staining of the membrane.
Commonly used secondary antibodies are conjugated with horseradish peroxidase (HRP) or alkaline phosphatase (ALP). HRP-conjugated secondary antibodies are most sensitive compared with the latter.
Various methods for protein detection on Western blots are available, including colorimetric and chemiluminescent reagents for horseradish peroxidase and alkaline phosphatase.
See a list of recipe calculators for various Western blotting buffers and blocking solutions.
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