There are several different types of agaroses commercially available, each with unique properties suited for specific applications. However, a common question that comes up is what is the difference between a standard molecular biology (MB) grade agarose and agarose LE (low electroendosmosis)?

The difference between agarose LE and a standard agarose is the level of electroendosmosis (EEO). Agarose LE stands for low electroendosmosis, making it well suited for PCR analysis and preparative electrophoresis. General purpose agarose, on the other hand, is used for routine electrophoresis.

For quick clarification, preparative electrophoresis refers to the purification of nucleic acids from an agarose gel.

Agarose gel band extraction as a preparative technique - preparative agarose electrophoresis

Figure 1. Example of preparative nucleic acid electrophoresis. DNA band being excised from an agarose gel. Purified DNA can be used in other downstream applications such as ligation.


The example in figure 1, where a DNA band is being extracted for purification, demonstrates how running an agarose gel works as a preparation step for DNA purification and further applications.

While preparative electrophoresis is different from general analytical work, researchers can and often do both – running an agarose gel for analysis and then purifying nucleic acids for PCR, modification, ligation and other techniques.

While knowing the difference between agarose LE and multipurpose molecular biology grade agarose (general purpose agarose) is helpful, you might have additional questions such as:

  • What is electroendosmosis?
  • Why is agarose with low electroendosmosis important?
  • When would I choose agarose LE, and when would I choose multipurpose agarose?
  • Are there times when a higher electroendosmosis is necessary?
  • What are the other types of agaroses out there?

In this article, we’ll take you question by question so you can be informed and confident when browsing agaroses and carrying out your experiment.


Article Contents:

What is electroendosmosis?

Let’s break this down a little bit:

Why is agarose with low electroendosmosis important?

The difference between agarose and agarose LE: digging deeper into the science

When would I choose agarose LE and when would I choose a multipurpose agarose?

Agarose choice based on what is being electrophoresed:

Nucleic acid electrophoresis for routine gels vs. analysis and downstream applications:

Choosing agarose based on approximate nucleic acid fragment size and gel concentration:

Are there times when a higher electroendosmosis agarose is necessary?

What are the different types of agaroses available?

GoldBio agarose & electrophoresis products

References



What is electroendosmosis in electrophoresis?

Before discussing the deeper differences between agarose LE and other agaroses, it is really important to explain what electroendosmosis means.

During electrophoresis, you have a porous agarose gel in buffer. When an electrical current is run, the fluid and DNA move through the pores of the gel. This is important when framing the explanation of electroendosmosis in electrophoresis applications.

Electroendosmosis is the movement of water and the contents within through a porous material when under the influence of an electric charge - positive and negative ionic charges caused by an electric current.


Let’s break this down a little bit:

At a molecular level, an agarose gel is a porous (figure 2), mesh-like material that researchers run nucleic acids through in order to distinguish fragments based on molecular size.

Rendering of the molecular structure of agarose, showing its porous nature.

Figure 2. Rendering of the molecular structure of agarose, showing its porous nature.


When performing electrophoresis, the gel is put into a rig where an electric current runs. On one side of the rig is a positive side (anode). On the other side is negative (cathode). The electrical current influences the movement of DNA, which has a slight negative charge.

Aside from DNA, other components can become ionized or carry slight charges, including buffer and even the sulfates (SO42-) bound to the agarose matrix.

Illustration of SO42- carrying a negative charge and bound to the agarose matrix. Sulfate content can impact electroendosmosis. SO42- bound to the agarose matrix is immobile; however, due to its negative charge, SO42- is still attracted to the anode (positive side).

Figure 3. Illustration of SO42- carrying a negative charge and bound to the agarose matrix. Sulfate content can impact electroendosmosis. SO42- bound to the agarose matrix is immobile; however, due to its negative charge, SO42- is still attracted to the anode (positive side).


Meanwhile, water in the buffer is hydrolyzed (H+ + OH-) during electrophoresis. The positive ions are attracted to the cathode side (negative).

Unlike the negative ions fixed within the gel matrix, the positive ions can migrate, creating resistance or a counterflow against the flow of DNA and other negatively charged particles.

Electroendosmosis in an agarose gel during electrophoresis. Positive ions within the buffer create resistance that can affect DNA mobility.

Figure 4. Electroendosmosis in an agarose gel during electrophoresis. Positive ions within the buffer create resistance that can affect DNA mobility.


From figure 4, we can see negatively charged DNA fragments as well as bound SO42- are attracted to the anode. But hydrolyzed water and other positive ions in buffer flow toward the cathode, causing a counterflow.

Imagine floating on an innertube down a river, traveling with the flow of the water when a motorboat from the opposite direction travels against the current. Its wake creates a counterflow that will start to impact your movement down the river.

This analogy might be a helpful way to understand the resistance and impact of electroendosmosis.



Why is agarose with low electroendosmosis important?

Varying levels of electroendosmosis matter for different methods in molecular biology.

Low EEO agarose is a more purified product with lower sulfate content. Sulfates are a contributor to the electroendosmosis phenomenon because of their negative charge.

When it comes to nucleic acid purification and analysis, agarose with low electroendosmosis (low EEO) allows better mobility, separation, and resolution because a lower EEO reduces counterflow resistance during electrophoresis.

Agarose LE (low EEO agarose) works for fragments larger than 1 kb and can work in agarose concentrations between 0.8% - 2.0%.

Additionally, agarose LE is ideal for blotting techniques.


The difference between agarose and agarose LE: digging deeper into the science

The difference between agarose LE and general-purpose agarose is the degree of electroendosmosis during electrophoresis. LE agarose is low electroendosmosis agarose, a property allowing more uniformed fragment movement with less hindrance in the gel.

When it comes to nucleic acid analysis or preparative electrophoresis, maintaining a more consistent movement through the gel is important.

Low EEO agarose have lower sulfate content, which reduces tension and counterflow. By reducing the amount of counterflow working against your nucleic acids, nucleic acid mobility and resolution increase and band distortion decreases.

These factors optimize electrophoresis, making it much better to use for analysis and preparative work.

Below, table 1 shows the typical EEO ranges for standard agarose, low EEO agarose, and high EEO agarose.


Table 1. Electroendosmosis ranges between low EEO agarose, standard agarose, and high EEO agarose.

Properties

Agarose LE

Agarose

Agarose HE

Typical EEO Range

0.07-0.13

0.12-0.15

0.23-0.27


One of the challenges when shopping for a low EEO agarose or choosing between a standard agarose and a low electroendosmosis agarose is that the property of LE, or Low EEO is not always labeled directly.

What we mean is that you might be looking at a product labeled Molecular Biology Grade Agarose. However, based on the product description or specifications, the characteristic of low EEO is not in an obvious place on the label or within the product specifications.

Examples of how agarose bottles might be labeled. EEO may be explicitly stated or must be found within the product specifications either on the label or the supplier’s website.

Figure 5. Examples of how agarose bottles might be labeled. EEO may be explicitly stated or must be found within the product specifications either on the label or the supplier’s website.


If the electroendosmosis properties are important to your experiment, we recommend you look closely at the product description.

In some cases, the agarose you are looking at will be a general-purpose agarose. Some specifications or descriptions might note lower or higher electroendosmosis.

Bottle A in figure 5 is an example of a bottle explicitly labeled for being a low EEO agarose.

Bottle B does not have LE or Low EEO in the product name, but the EEO content shown in the specifications are considered low.

Bottle C is an example of a multipurpose type of agarose. Based on the stated EEO specification, the EEO is slightly higher.

While the images in the figure list EEO on the bottle, whether the EEO specification shows up on the label depends on the supplier.

If there is ever a question, we emphasize looking closely at the supplier’s product page and product description.

GoldBio carries low EEO agarose, which is labeled as Agarose LE (≤0.12). This agarose is also molecular biology grade, meaning it is equal to ultra-pure chemicals and is ideal for molecular biology applications.



When would I choose agarose LE and when would I choose a multipurpose agarose?

Deciding whether to use a lower, standard, medium, or higher EEO agarose depends on what is being electrophoresed, downstream applications, fragment size, and gel concentrations.

Because low EEO agarose is a more purified agarose, it also reduces the effects of contaminating contents when purified products are used in downstream applications.

When considering what type of agarose to use, a few questions to consider are:

  • What is electrophoresis being performed on: nucleic acids or proteins?
  • Is this a routine gel, or will I do analysis or downstream applications?
  • What is the approximate fragment size I’m working with, and what concentration is my gel?


Agarose choice based on what is being electrophoresed:

For nucleic acids (DNA and RNA), it’s best to stick with a standard or low EEO agarose. In the section that follows, we’ll explain deeper when to choose a multipurpose agarose or an LE agarose.

However, table 2 provides a quick lookup to help you decide between a multipurpose agarose (MP) or agarose LE.

When using electrophoresis to separate certain proteins such as serum proteins or for immunoelectrophoresis, agarose with higher electroendosmosis should be used.


Table 2. Agarose choice based on what is being electrophoresed.

Properties

Agarose MP

Agarose LE

Agarose ME

Agarose HE

Nucleic Acids for Routine Gels

X

X



Nucleic Acids for Downstream Applications


X



Serum Proteins



X

X


Key: Agarose MP (multipurpose or general-purpose agarose), Agarose LE (low EEO agarose), Agarose ME (medium EEO Agarose), Agarose HE (High EEO agarose)



Nucleic acid electrophoresis for routine gels vs. analysis and downstream applications:

When it comes to choosing between a standard (multipurpose) agarose or agarose LE (low EEO) during DNA or RNA electrophoresis, the choice comes down to your goals.

Choose a general-purpose (multipurpose) agarose for routine nucleic acid electrophoresis. For analysis, blotting, or other downstream applications such as ligation, purification, and blotting, use a low electroendosmosis (low EEO) agarose.

Low EEO agarose allows better resolution and band separation as well as has lessl contaminants such as SO42-, which makes it better suited for downstream work.



Choosing agarose based on approximate nucleic acid fragment size and gel concentration:

There is an important relationship between DNA fragment size and agarose gel concentration. Smaller fragments require a higher agarose gel concentration, while larger fragments need a lower agarose gel concentration.


Table 3. Recommended agarose concentrations based on DNA size.

Concentration (%)

DNA Size (bp)

0.5

1,000 – 25,000

0.8

800 – 12,000

1.0

500 – 10,000

1.2

400 – 7,500

1.5

200 – 3,000

2.0

50 – 1,500


There is also a relationship between electroendosmosis and agarose gel concentration. As the concentration of an agarose gel increases, electroendosmosis also increases because this consequently increase in the concentration of ionized functional groups that contribute to electroendosmosis.

This is an important consideration when choosing whether you need a standard agarose or agarose LE.



Are there times when a higher electroendosmosis agarose is necessary?

Agarose with high electroendosmosis and medium electroendosmosis is necessary for certain protein applications such as electrophoresis of serum proteins. They can also be used for immunoelectrophoresis and counterimmunoelectrophoresis.

With an increased tension from electroendosmotic counterflow, different protein samples will move differently through the gel. This will help obtain better protein separation.

Serum proteins are a particularly good example because higher electroendosmosis allows gamma globulins from serum to have much better separation.



What are the different types of agaroses available?

Common agaroses include general purpose (multi-purpose) agarose; high, medium, and low EEO agarose; pulse field gel electrophoresis agarose; high-resolution agarose; low melt agarose; etc.

List of common types of agaroses:

  • General or multipurpose agaroses (agarose MP)
  • Agarose LE (low EEO)
  • Agarose ME (medium EEO)
  • Agarose HE (high EEO)
  • Pulse field gel electrophoresis agarose
  • High-resolution agarose

In addition to this list, there are different grade agaroses available such as molecular biology grade agarose options and genetic quality tested.

Different suppliers have also developed different varieties and specialty agaroses for unique needs.



GoldBio agarose & electrophoresis products


References

Guo, Y., Li, X., & Fang, Y. (1998). The effects of electroendosmosis in agarose electrophoresis. Electrophoresis, 19(8‐9), 1311-1313.

LabCE. (n.d.). Electroendosmosis. Electroendosmosis - LabCE.com, Laboratory Continuing Education. Retrieved July 11, 2022, from https://www.labce.com/spg206971_electroendosmosis....

Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. JoVE (Journal of Visualized Experiments), (62), e3923.

PRK LECTURER SERIES. (2020, December 7). Electrophoretic mobility I factors affecting electrophoretic mobility I electroendosmosis. YouTube. Retrieved July 11, 2022, from

Rizzi, C., & Carraro, U. (1991). Electroendosmotic preparative gel electrophoresis and peptide mapping of slow and three fast myosin heavy chains. Basic Appl Myol, 1, 43-53.

Upcroft, P., & Upcroft, J. A. (1993). Comparison of properties of agarose for electrophoresis of DNA. Journal of Chromatography B: Biomedical Sciences and Applications, 618(1-2), 79-93.

Westermeier, R. (2001). Gel electrophoresis. e LS.