Western Immunoblot Introduction
Western blotting, also known as immunoblotting, is a technique used to detect the presence and molecular weight of specific proteins. Western blot utilizes the specificity of antibodies to detect a certain protein within a sample such as tissue or cell lysate. This protocol provides a set of initial conditions for analysis of cell lysates for Western blot. Further optimization may be required for individual samples or analytes. Follow manufacturer's protocols for specific reagents when applicable.
Please read the following Western blot cell lysate protocol in its entirety before beginning. View protocol for Electrophoresis and Protein Transfer.
Western Blot Cell Lysate Preparation
- Prepare total cell lysates by solubilizing cells in an appropriate sample buffer, such as 2X SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenol blue), at approximately 2x106-1x107 cells per mL.
- Heat the extracts in a boiling water bath for 5 minutes
- Sonicate for 5-10 seconds 3-4 times
- Prepare the following solutions:
- Transfer the electrophoresed proteins to a PVDF membrane and incubate for 1 hour at room temperature in Blocking Solution.
- Incubate the PVDF membrane overnight at 4°C in Antibody Solution containing primary antibody.
- Wash the PVDF membrane at room temperature for 30-60 minutes with 5 or more changes of Blotting Buffer.
- Incubate the membrane for 1 hour at room temperature in Antibody Solution containing appropriate dilution of HRP-conjugated secondary antibody.
- Wash the membrane for 30-60 minutes with 5 or more changes of Blotting Buffer.
- Detect with Chemiluminescent Detection Substrate.
- Expose to film and develop image.
|Blotting Buffer||Blocking Solution||Antibody Solution|
|25 mM Tris, pH 7.4
0.15 M NaCl
0.1% Tween® 20
|2-5% nonfat dry milk in Blotting Buffer
Adjust pH to 7.4
|1-5% nonfat dry milk in Blotting Buffer
Adjust pH to 7.4
Note: The above buffers are recommended as a starting point. Further optimization may be required depending on your cell lysate and protein of interest. View all our recommended Western Blot Buffer Groups.
Note: if the primary antibody is conjugated with HRP, this step should be omitted and proceed directly to step 6
Optimization of Immunoblotting:
When running Western blot with cell lysates, multiple parameters may need to be optimized for antibody detection of endogenous protein levels. The objective in optimizing Western blotting conditions is to maximize signal strength and minimize non-specific bands and background noise. The variables with the most significant impact are listed below. Optimization may be done as an initial checkerboard screen where multiple conditions are applied in a single experiment or sequentially, changing one set of parameters at a time and optimizing conditions over several blots.
Recommended starting conditions:
- Primary antibody concentration. 0.1-0.5 μg/mL. Adjust antibody concentration from 0.05 up to 2.0 μg/mL to obtain desired signal strength and low background.
- Sample concentration. 10-20 μL of cell lysates at 1x107 cells per mL. (This is typically equivalent to 15-30 μg of total protein). Adjust up or down to obtain desired signal strength and low background.
- Blocking buffer. Start with 5% nonfat dry milk for block, and 2% nonfat dry milk for primary and secondary antibody dilution. Adjust concentration of milk up or down to obtain desired signal strength and low background. If the intensity of the target band is still too low, but background is not a problem, 1% BSA can be used as the blocking component.
- View our recommended Western Blot Buffer Groups.
- NaCl concentration. Recommended concentration is 0.15M NaCl. Increasing the salt concentration in all buffers to 0.5M NaCl will reduce background. Note: high salt can also reduce signal strength of the target protein.
Tween is a registered trademark of ICI Americas.
Western Blot Data Example