Poor separation of protein targets is one of the most common and frustrating issues with associated with Western blotting. To troubleshoot the problem, we must first understand how protein separation by SDS-PAGE works. SDS-PAGE stands for sodium dodecyl sulfate (SDS) Polyacrylamide gel electrophoresis (PAGE). It is the universal first step in denaturing Western blots. The goal of SDS-PAGE is to separate proteins by their molecular weight.
How do you separate proteins using SDS-PAGE?
Polyacrylamide gels form a crosslinked, mesh-like matrix through which proteins migrate with the application of current. The matrix functions like a sieve, allowing smaller, low molecular weight proteins to migrate quickly through the gel, while large, high molecular weight proteins tend to progress more slowly. When the electrophoresis is finished, proteins in each lane will be separated into a continuum, with the highest molecular weight proteins remaining near the loading site and the lowest molecular weight proteins reaching the opposite end of the gel.
An important caveat to this rule is this: proteins only separate exclusively by size if they have been denatured prior to being run on a gel. All proteins have inherent tertiary structures related to their biological functions. Such structures confound the process of separation by molecular weight alone. Proteins of any size with globular structure tend to migrate slower through the gel matrix, giving them a similar migration pattern to proteins of higher molecular weights. Such proteins can easily be misidentified if identification is based only on molecular weight.
Because of this phenomenon, it’s important you remember to denature your protein samples prior to separation by SDS-PAGE to remove higher levels of structure. This step also allows the proteins to be separated almost exclusively by molecular weight as they migrate through the gel matrix. SDS detergent, the denaturing agent DTT, and boiling are all used to disrupt higher structure and linearize proteins.
Many proteins also have an inherent negative or positive charge, which can disrupt uniform migration driven by electrical current, as in gel electrophoresis. SDS used for denaturing also serves to coat the proteins with an overall negative charge, allowing the proteins to migrate in response to electric current without influence from their natural charge. As denaturing reduces the confounding effects of protein structure on migration, so uniformly charging reduces the effects of native charge.
6 Suggestions to How to Improve Separation of Bands by SDS-PAGE
With an understanding of how SDS-PAGE facilitates separation of proteins by molecular weight, it is possible to identify areas of the process that may contribute to band separation issues. Here are 6 ways you can start troubleshooting:
1. Ensure proper sample preparation
If the proteins in a sample are not denatured properly, they will not migrate through the gel as expected. You have to ensure the amount of SDS and DTT used to denature the sample is appropriate. Try slightly increasing the boiling time to make sure all samples are completely denatured (although boiling too long can degrade proteins, so finding an appropriate boiling time is key). About 5 minutes at 98°C is common for samples prepared with denaturing loading buffer.
After boiling, place the samples immediately on ice to prevent gradual cooling prior to loading. Allowing the samples to naturally return to room temperature will undo the denaturing step, as most proteins will renature if allowed to cool slowly.
2. Load the appropriate amount of protein sample
Loading an excess of protein per well can cause the proteins to aggregate during electrophoresis, preventing them from separating by size and causing them to run together. This can result in clustered bands that cannot be individually defined. Additionally, excess protein can bleed into neighboring lanes, preventing sharp lane distinctions.
For best results, use the minimum amount of protein or cell lysate required for downstream detection. It is always necessary to validate each protein-antibody pair and it may take multiple tests to determine the optimal amount to load for each protein of interest. On the other hand, loading too little protein will result in faint or undetectable bands.
3. Make sure your polyacrylamide gel is fully polymerized
The polymerization of the polyacrylamide gel is important for efficient electrophoresis. Make sure the gel has had enough time to completely polymerize before using in PAGE. Incomplete polymerization is often caused by forgetting to include key ingredients, especially TEMED. Double check you have added all the ingredients, that they’re fresh, and added in the correct concentrations.
If your budget allows, purchasing pre-made polyacrylamide gels can be a simple solution to these polymerization issues.
4. Change the polyacrylamide gel percentage
The percentage of polyacrylamide used in a gel affects the number and size of pores in the matrix. Gels made with high amounts of polyacrylamide have small, tight gel matrixes; those made with low amounts of polyacrylamide have larger, more spacious matrixes. Proteins of all sizes migrate more quickly through lower percentage polyacrylamide gels and more slowly through higher percentage gels. Because of this, gel percentage should be chosen based on the size of the protein of interest.
High molecular weight proteins require gels with a low percentage of polyacrylamide. If the matrix is too small, these large proteins will be not be able to migrate efficiently and will stay grouped together near the loading site. Similarly, low molecular weight proteins require high percentage gels for effective separation. In a low percentage gel, small proteins tend to migrate too quickly. The open gel matrix allows small proteins of a range of molecular weights to migrate together, resulting in poor band resolution.
Because these pores are the reason certain proteins migrate through the gel, using a gel made with the appropriate percentage of polyacrylamide for the protein of interest is very important. The higher the percentage of polyacrylamide, the harder it is for proteins to move through the gel. This can be helpful if you are wanting to separate proteins that are small. If you are using a higher percentage gel on larger proteins, they cannot fully separate because they won’t be able to move through the gel.
If proteins of interest are relatively small and are not spreading, try using a higher percentage of polyacrylamide gel. If the proteins of interest are larger and are not spreading, try a lower percentage gel.
ADDITIONAL RESOURCE: Bendy Bands and What to do with Them
5. Use fresh buffers
The buffers used for running SDS-PAGE require very specific salt concentrations and other components to allow the current flow properly and the proteins remain denatured. Protein separation can be hindered by overused or improperly formulated buffers. It is good practice to make fresh buffers before each run or as frequently as possible if large volumes are used regularly
6. Adjust the electrophoresis parameters
Finally, gel temperature is affected by the amount of current applied to the gel and can affect the migration patterns of proteins. Generally, the gel should be prevented from getting too hot. A simple way to address this is to run the gel at a lower voltage for longer time. With the Azure Aqua Transfer Cell, you’re able to perform quick, 1 hour transfers or transfers overnight at lower voltages. Alternatively, the gel can be kept cool by placing a compatible ice pack in the buffer chamber or by placing the entire gel apparatus in a cold room while it runs.
We want to see your Western blots succeed! Band separation is key for successful SDS-PAGE and downstream applications, such as Western blotting. Issues with band separation can occur, but the six troubleshooting tips provided here will help you solve the issues and return to collecting data quickly.
Here at Azure Biosystems, we are dedicated to bringing you quality products that will help you run flawless experiments and obtain quality data every time. Check out this page to make sure you have everything you need to run a successful Western blot. Be sure to use the form on this page to ask for help!
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