Overview: Electrophoresis Buffers

VIEW ELECTROPHORESIS BUFFERS

Gel electrophoresis is a foundational laboratory technique used to separate biomolecules (DNA, RNA, proteins)[1-3], based on differences in size, charge, and shape. It is widely used in molecular biology and biochemistry for applications such as analyzing nucleic acids, characterizing proteins, detecting mutations or post-translational modifications, and more.

 

In a typical workflow, a porous gel (agarose or polyacrylamide) is cast in a buffer, samples are prepared with loading buffers, and an electric field is applied to drive migration through the gel. Smaller or more highly charged molecules generally migrate faster than larger or less charged species.

Electrophoresis Buffers Overview Page

 

Image 1: Polyacrylamide gel electrophoresis (PAGE) is one example of gel electrophoresis that can be used to separate protein samples. Not to be confused with agarose gel electrophoresis, PAGE is presented in a vertical gel, where results can be visualized.

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What is an Electrophoresis Buffer?

An electrophoresis buffer is a solution that provides:

  1. Ionic strength and conductivity, allowing current flow through the gel environment.
  2. pH control / buffering capacity, ensuring stable pH during migration so that the molecules retain predictable charge states.
  3. Compatibility with the molecules of interest, not interfering with the biomolecules or gel chemistry.

In electrophoresis workflows, multiple types of buffers are used at different stages (casting, running, transfer, etc.), each optimized for its role.

Common Electrophoresis Buffer Types & Their Roles

Below is a table summarizing the main buffer classes in electrophoresis and what they are used for:

Buffer Type Purpose / Role Typical Components & Notes
Gel Casting Buffer To prepare the gel matrix (agarose or polyacrylamide) Contains the buffering ions (e.g. TAE or TBE for agarose, Tris-HCl for PAGE) and provides stable pH and ionic environment during polymerization and later migration
Sample / Loading Buffer To prepare the biomolecule sample before loading May include denaturants (e.g. SDS), reducing agents (e.g. DTT or β-mercaptoethanol), tracking dyes (e.g. bromophenol blue), glycerol or other density agent to help loading
Running / Electrophoresis Buffer To fill the tank / chamber and ensure conduction during migration Usually, the same or closely matched buffer system as the gel (to avoid pH/ionic discontinuities)
Transfer Buffer To move molecules from gel into membranes (blotting) For proteins: often Tris-Glycine (plus methanol). For nucleic acids: Tris-Borate, or other suitable buffer systems. Methanol (10–20%) may be added to assist binding of proteins to membrane
Blocking Buffer To block nonspecific binding sites on a membrane after transfer Contains a protein (e.g. BSA, non-fat dry milk) or other blocking agent to reduce background in immunodetection
Stripping Buffer To remove primary/secondary antibodies from the membrane for reprobing Often low pH (e.g. glycine-HCl) to disrupt antibody–antigen binding

In practice, which buffer formulations you use depends on the analyte (DNA, RNA, protein) and the downstream detection or processing steps.

Electrophoresis Overview

Applications of Electrophoresis Buffers

Electrophoresis buffers enable a wide range of common laboratory techniques, including:

  • DNA / RNA electrophoresis & Southern / Northern blotting: using agarose gels and buffer systems like TAE, TBE, Tris-Borate, etc.
  • Protein separation (PAGE / SDS-PAGE): electrophoresis of proteins before transfer to membranes for Western blotting or mass spectrometry.
  • Protein blotting (Western or Eastern blotting): buffer systems designed for optimal transfer of proteins from gel to membrane.
  • Nucleic acid transfer / blotting (Southern, Northern): buffer systems for transfer of DNA or RNA onto membranes for probe hybridization.
  • Membrane immunoassays / immunodetection workflows: e.g. after transfer, using blocking buffers and antibody incubation steps.

Because buffers are integral to each step, choosing the right buffer, or customizing one, is critical to ensure clean separation, minimal artifacts, and compatibility with downstream detection.

 

Boston BioProducts offers both off-the-shelf electrophoresis buffers and the option for custom buffer formulation / manufacturing to meet specific experimental needs.

Practical Tips & Best Practices

  • Match buffer systems: Use the same ionic system (or very compatible ones) for gel casting and running buffers to maintain steady pH and field strength.
  • Maintain pH stability: Changes in pH can alter molecule charge states, migration behavior, and resolution quality.
  • Fresh reagents — Reducing agents, dyes, or other labile components degrade over time. Use freshly prepared or aliquoted solutions.
  • Avoid salt overloads / interfering substances — High salt or incompatible additives in your sample will distort fields or cause smearing.
  • Optimize buffer concentration / strength — Too low ionic strength leads to poor conductivity and slow runs; too high can lead to excessive heat or distortion.
  • Temperature control / cooling — For long electrophoreses, overheating can degrade resolution; use cooling or lower voltage if needed.
  • Methanol / additives in transfer buffer — In protein transfer, methanol helps binding to membrane but may reduce gel porosity; balance accordingly.
  • Blocking and stripping — After transfer, ensure blocking buffer covers all nonspecific sites, and for reprobing, stripping buffer should be gentle but effective.

Frequently Asked Questions (FAQs)

Choose based on your analyte and resolution needs. For DNA agarose gels, TAE (Tris-Acetate-EDTA) and TBE (Tris-Borate-EDTA) are common. TAE gives faster migration but lower buffering capacity; TBE gives sharper bands and better resolution for small fragments. For proteins and blotting, Tris-Glycine or Tris buffers are standard.

Yes — in fact, using the same buffer for both (or highly compatible ones) is preferred to maintain consistency in ionic strength and pH during electrophoresis.

Not necessarily. Reducing agents (like DTT or β-mercaptoethanol) break disulfide bonds, which is desired when you want full denaturation and analysis of monomeric proteins. But if your goal is to preserve disulfide bonds (e.g. studying quaternary structure or conformational epitopes), you may use non-reducing buffers.

Methanol (typically 10–20%) helps strip SDS from proteins and encourages binding to the membrane (particularly nitrocellulose). However, too much methanol may reduce gel pore sizes and hamper transfer, so optimize concentration.

For short-term use, store at recommended temperatures. For buffers containing reduced agents or other labile components, aliquot and store components separately if possible. Avoid repeated freeze-thaw cycles.

Potential causes include overloaded lanes, salt contamination, degraded reagents, improper buffer pH or ionic strength, or overheating. Troubleshoot by diluting your sample, desalting or cleaning buffer components, replacing reagents, and ensuring proper voltage/temperature control.