Electrophoresis 101: the Difference between Running and Transfer Buffer

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SDS-PAGE vs. Western blotting- what's the difference?

SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a commonly used technique for the separation of proteins based on their size. In SDS-PAGE, smaller proteins migrating more quickly through the gel inside of a transfer cell. The percentage of polyacrylamide in the gel determines how easily proteins of various sizes can move through the gel, with higher percentage gels having a tighter gel matrix better for resolving smaller proteins.

Western blotting is a commonly used method for detecting and analyzing the separated proteins, after being separated using SDS-PAGE.

The role of buffers in Western blotting

An important aspect of Western blotting is the use of buffers, which play a crucial role in the success of the experiment. Two types of buffers are commonly used in Western blotting: running buffer and transfer buffer. Understanding the difference (Table 1) between the two and what their specific functions are is crucial to a successful Western blotting experiment.

Table of contents

What's the difference between running buffer and transfer buffer?

For conducting nucleic acid or protein electrophoretic separations, running buffer is an essential, as it provides the required solutions.

Transfer buffer is a solution used in the process of Western blotting. It is used to transfer the separated proteins from the gel to a solid support, such as a nitrocellulose or PVDF (Polyvinylidene Fluoride) membrane. The transfer step allows for the detection and analysis of specific proteins of interest. Keep reading for ingredients and guidelines on how to use each type of buffer.

Table 1. The difference between running and transfer buffer

What it isWhen it's used
Running bufferRunning buffer is a solution used in electrophoresis, specifically in SDS-PAGE.Running buffer's main purpose is to create an electric field that allows for the movement of proteins through the gel during the electrophoresis process.
Transfer bufferTransfer buffer serves as a conductive medium to facilitate the transfer process. It is designed to preserve a consistent pH level, aiding the elution of proteins from the gel, as well as their binding to the membrane.Transfer buffer is used in the process of Western blotting and electrophoretic transfer.

How do you make running buffer?

Running buffer is made up of a mixture of SDS and a salt, such as Tris-HCl, which helps to maintain the pH and conductivity of the solution. The concentration of SDS in the running buffer is usually around 1% and helps to uniformly denature and negatively charge the proteins, allowing for their separation based on size and not charge1.

To make the running buffer, mix the following ingredients in distilled water:

  • 10X

    SDS running buffer concentrate (e.g. 1 M Tris-HCI, 0.1 M SDS)
  • 50 mM

    Tris-HCI (pH 8.3)
  • 1%

  • Distilled water to make the final volume of the solution

pH variations can affect the separation of proteins, which is why the pH of the running buffer should be checked and adjusted to 8.3 before use. The running buffer should be pre-heated to 70-75°C to solubilize the SDS when first making it, before cooling it down to room temperature.

Can I reuse running buffer?

Reusing running buffer is generally not recommended because it can affect the accuracy of protein separation in SDS-PAGE. Running buffer becomes contaminated with protein fragments and other debris after each electrophoresis run, which can interfere with subsequent runs and lead to unreliable results. Additionally, the SDS and other components in the buffer can degrade over time, affecting its effectiveness.

For these reasons, we recommend preparing fresh running buffer for each experiment to ensure your results are accurate and consistent. It is possible to prepare and store a larger batch for a short period of time (e.g. a few days) in a refrigerated environment; however, you should still replace it regularly to save time.

How do you make transfer buffer?

Before jumping into mixing your own, you should know that transfer buffer can easily be purchased. The Azure transfer buffer is a proprietary transfer buffer that provides improved detection of low-abundance and post-translationally modified proteins. We offers free samples of it if you find yourself wanting to eliminate the time it takes to mix your own. High-efficiency protein transfer and increased protein retention on the membrane add up to more sensitive Western blots.

However, the answer to how to make transfer buffer is simple: it typically contains a combination of salt, such as Tris-HCl, and a reducing agent, such as methanol, to help maintain the stability of the proteins during transfer by dissociating SDS from the proteins. Methanol is also important for helping the proteins adhere to the membrane. Unlike running buffer, transfer buffer does not contain SDS, as its role is to preserve the proteins and not to denature them. The pH of the transfer buffer is usually around 7-8 to ensure optimal protein transfer and stability.

Transfer buffer should be prepared fresh for each experiment, because its effectiveness can be affected by long-term storage or repeated use. To ensure consistency and reliability, purchasing transfer buffer is a great idea. Using pre-made transfer buffer provides improved detection of low-abundance and post-translationally modified proteins. The combination of high-efficiency protein transfer and increased protein retention on the membrane amount to more sensitive Western blots. Using pre-made transfer buffer allows you to transfer proteins in less than 20 minutes vs. several hours required with traditional, homemade transfer buffers.

Mix your own transfer buffer using this recipe:

  • 50 nm

    Tris-HCI (pH 8.0)
  • 20%

  • 0.1%

  • Distilled water to make the final volume of the solution

For additional buffer recipes, check out the app note Wet or Dry?

How many times can transfer buffer be used?

The number of times transfer buffer can be reused depends on several factors, including the composition of the buffer,  the size and stability of the proteins being transferred, and the conditions of storage and use. In general, reuse of transfer buffer is not recommended, as the efficiency of protein transfer can decrease with repeated use.

Proteins can become trapped in the gel or in the pores of the transfer membrane, reducing the overall efficiency of transfer. Additionally, the reducing agent in the transfer buffer can degrade over time, affecting its ability to preserve the proteins during transfer resulting in inaccurate results. The presence of contaminants or impurities in the buffer can also reduce its effectiveness.

For these reasons, we suggest you prepare fresh transfer buffer for each experiment to ensure your results are accurate and reliable. If cost or availability is a concern, transfer buffer can be stored for a short period of time (e.g., a few days) in a refrigerated environment, but it should still be replaced regularly to ensure optimal results.

Quick, 1-hour or transfers overnight at lower voltages

The Azure Aqua Transfer Cell is able to keep gels cool through a compatible ice pack in the buffer chamber. You can also place the entire gel apparatus in a cold room while it runs.
Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside an Azure Aqua Vertical Gel Running System

We hope after reading this article you are able to easily distinguish the difference between transfer and running buffers. If you still have questions regarding the steps of Western blotting, fill out the form below and someone from our team will assist. Additional resources on SDS page can be found below as well. Cheers!

Additional resources for gel electrophoresis and SDS-PAGE:

  1. Gavini K, Parameshwaran K. Western Blot. [Updated 2022 Apr 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK542290/

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