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Western Blot Experimental Design Tips

When planning your western blot experiment, there are a few things to keep in mind.

Make sure to include the appropriate positive and negative controls. To find more information on the controls Cell Signaling Technology (CST) uses when testing our antibodies, please refer to the antibody’s webpage or the Control Treatments by Target table. We have also found that BioGPS and The Human Protein Atlas are excellent online resources for information about a given gene product's relative abundance in different cell types and tissues.

Detection of post-translationally modified proteins may require specific treatments. CST® product webpages often provide examples of treatments that activate a particular post-translational modification in one or more cell models. Moreover, the Control Treatments by Target table lists validated positive controls for most modification-specific antibodies and any control extracts that are available for purchase from CST. PhosphoSitePlus® is also an excellent online resource that provides a quick overview of the modified residues on a given target, their functional significance, and published references for treatments that modulate a given post-translational modification in specific cell models.

Select the right gel for optimal resolution of your protein. CST validates the majority of our antibodies for western immunoblotting using samples resolved on Tris-Glycine gels. Because they allow for the resolution of targets over a broad range of molecular weights, we most often use 4-20% Tris-Glycine gradient gels. However, single percentage gels are easier to prepare in the lab and can give superior resolution over a more limited molecular weight. The general rule is the smaller the molecular weight, the higher the percentage of gel that needs to be used and vice versa. We recommend the following gels based on the target protein's molecular weight.

Gel Type Protein Molecular Weight
3-8% Tris-Acetate* > 200 kDa
4-20% Tris-Glycine 10-200 kDa
7.5% Tris-Glycine 80-200 kDa
10% Tris-Glycine 50-80 kDa
12% Tris-Glycine 30-60 kDa
16% Tris-Glycine 12% Tris-Glycine < 30 kDa

*Tris-Acetate gels used with Tris-Acetate SDS buffer run at a neutral pH. As a result, Tris-Acetate gels can be run for extended periods of time, even overnight, without proteins migrating out of the gel. Since larger proteins require longer run times to achieve separation, Tris-Acetate gels serve as a better option than Tris-Glycine for proteins with a high molecular weight.

Choose a secondary antibody that is compatible with your primary antibody. All unconjugated CST antibodies are validated in house for western immunoblotting using our anti-IgG, HRP antibodies. Rabbits have only one IgG subtype, therefore the Anti-rabbit IgG, HRP-linked Antibody (#7074) is appropriate for use with all rabbit polyclonal and monoclonal antibodies. The Anti-mouse IgG, HRP-linked Antibody (#7076) is recommended for use with mouse monoclonal antibodies, as it recognizes all mouse IgG subtypes. Similarly, the anti-rat IgG, HRP-linked Antibody (#7077) is recommended for use with rat monoclonal antibodies, as it recognizes all rat IgG subtypes.

Follow the recommended protocol. CST antibodies are developed and optimized using the conditions outlined in the protocols listed on the product’s webpage. When using an antibody for the first time, use the recommended buffers and dilutions to obtain optimal results. Deviations from the recommended protocol may result in low signal or high background, resulting in the need to repeat experiments.

Considerations for Fluorescent Western Blotting

For optimal fluorescent western blotting conditions, we recommend following the CST fluorescent western blotting protocol closely. Some notable alterations to the chemiluminescent protocol include:

  • Omit Tween® 20 from the blocking buffer during the 1-hour blocking step. Inclusion of Tween® 20 in this step can affect detection with the antibody and lead to low signal.
  • Use the product-specific primary antibody incubation buffer recommended by CST or the LI-COR® TBS-based incubation buffer. Using other dilution buffers may lead to a weak or absent signal.
  • Prior to imaging, ensure the membrane is air-dried to improve overall detection.
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