This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
, No Gclids
Western Blot Optimization:
BLOCKING BUFFER OPTIMIZATION
Choosing the right blocking buffer is key to a clean successful Western blot. If you run into background or specificity issues and suspect it is the blocking that failed you, use this guide to decide what better options you may have.
Click for more optimization tipsBSA vs Non-Fat Dry Milk
The blocking step of Western blotting prevents nonspecific binding of the antibody to the membrane by saturating the membrane with nonreactive proteins. The most common blocking buffers include 3-5% bovine serum albumin (BSA) or nonfat dried milk in phosphate-buffered saline (PBS) solution or tris buffered saline (TBS) solution. Small amounts of the detergent Tween 20 can be added to blocking solutions and washing solutions to further reduce background staining.
Each Blot May Require Different Blocking Buffer
To obtain the best results from your western blot, you must decide which type of blocking buffer to use based on the detection system used and the antigen you’re trying to detect. For example, the phosphate in PBS interferes with alkaline phosphate detection systems, making TBS blocking buffer the better choice: Proper selection of blocking buffers is crucial for minimizing nonspecific binding and optimizing signal-to-noise ratios in your Western blot protocol.
Advantages and Disadvantages of Western Blot Buffers
| Buffer | Advantages | Disadvantages |
|---|---|---|
| BSA blocking buffer | -Compatible with all detection systems and antibodies | -Doesn’t inhibit nonspecific antibody binding |
| -Allows for higher sensitivity detection | -Less complete blocking | |
| -More expensive | ||
| Nonfat dry milk blocking buffer | -Inexpensive | -Incompatible with phospho-antibodies |
| -More complete blocking | -Incompatible with avidin/biotin detection systems | |
| -Can reduce nonspecific antibody binding | ||
| Boster TBS blocking buffer | -Least expensive | -Ships as a powder |
| -Compatible with all antibodies and detection systems | -Cannot be swapped into a different buffer (e.g. PBS for TBS) | |
| -Allows high sensitivity detection |
Related Pages
Western Blotting Assay Optimization Tips
Get to learn the concept behind our best practices on Western Blot optimization. Learn how to optimize every aspect of your experiment to yield the best results.
See MoreWestern Blotting Principle, How Western Blots Work
Learn the concept behind Western blotting. It is a technique that is used to detect specific proteins in the given sample. It usually involves two major processes, namely, SDS-polyacrylamide gel electrophoresis and protein blotting and testing.
See MoreOptimizing Western Blot Blocking
There are three factors to consider when choosing an appropriate blocking agent for your specific protocol: (Click the meme for blocking optimization tips!) We advise reviewing the data sheet for any recommended blocking agents because antibodies can...
See MoreWestern Blot Troubleshooting
These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer. Learn about Western Blot Principle Western Blot Sample Preparation Check out this...
See MoreWestern Blotting Antibody Concentration Optimization
Each Western blot experiment involves a unique antibody interacting with a unique sample under varying conditions. No single antibody or antigen concentration will work for every experiment. In order to obtain the most perfect Western blots, the antibody concentration needs to be optimized. The ideal antibody concentration is dependent on the concentration of antigen, the specificity and affinity of the antibody, and experimental conditions such as buffer composition.
See MoreELISA Blocking Optimization
There are a variety of blocking buffers, not one of which is ideal for every combination of plate type, assay format, and detection system. Every blocking buffer represents a compromise between reducing background and maintaining specificity. Use this guide to help decide which type of blocking buffer is best suited for your specific application.
See MoreWestern Blot Protocol
Learn A Stepwise Western Blotting Protocol From Reagent Preparation To Detection With Application Of BosterBio Reagents . Check Out Our ELISA Protocols To Learn How To Get The Best Results.
See MoreELISA Optimization Tips
Use this guide to help decide whether to use enzyme-linked secondary antibodies, or fluorescently labelled secondary antibodies as your detection system. ELISA Blocking Buffer Optimization There are a variety of blocking buffers, not one of which is ...
See More