Agarose

CAS Number: 9012-36-6 (product A9539)
CAS Number: 39346-81-1 (products A9414, A4018, A6560, A9045, A0701)
Synonyms: 3,6-Anhydro-α-L-galacto-β-D-galactan; FastLane agarose; Indubiose A4; NuSieve GTG; Odigose; Seakem; Sepharose

Agarose Structure

What is the difference between agar and agarose?

Agarose is a polysaccharide that is isolated from agar or agar-bearing marine algae (sea kelp). Agar is composed of two polysaccharides, agarose and agaropectin. While agar is a neutral gel, agaropectin is highly sulphated and does not form a gel. Structurally, agarose is a linear polymer consisting of alternating β-D-galactose and 3,6-anhydro-L-galactose units.4

Why is agarose used instead of agar?

While agar consists of a variety of polysaccharides including agarose, a sulphated galactan or porphyran, agarose is purified from agar, has lower sulphate content and is more neutral in charge. Higher impurities in the agar gel may retard the migration and separation of substances (such as DNA and proteins) and affect the biological activity of these substances if they are purified from this gel. Therefore, agarose gel with lesser impurities is preferred over agar gel.5

What is agarose used for?

As a gelling agent, agarose is used in:

  1. Electrophoresis: Agarose is used for a wide range of electrophoresis applications including separation of nucleic acids, immunoelectrophoresis (IEP) and isoelectric focusing. Unlike polyacrylamide, the consistency of the gels is more solid (but also less elastic).6
  2. Electrophoresis


  3. Immunodiffusion: Agarose is used as the solid gel matrix through which antibody or antigen molecules are allowed to diffuse to test for cross-reactivity. Ouchterlony is a specific double diffusion technique which tests for cross-reactivity of antigens or antibodies.8
  4. Immunodiffusion


  5. Solid Culture Media: Agarose is used to make gel plates or overlays for cells in tissue culture. It is also used to prepare solid or semi-solid media for the growth of bacteria or plant cells. Another use could be to cover virus-infected cells in plaque assays.
  6. Solid Culture Media


  7. Gel Chromatography, Affinity Chromatography, and Ion Exchange Chromatography: Agarose may be used to form the solid matrix in which reactive molecules are embedded to interact with a liquid phase passed over the solid matrix (either beaded and/or crosslinked) in chromatographic applications to isolate, purify, and identify a macromolecule.1
  8. Gel Chromatography


  9. Growth of protein crystals: Agarose can minimize the diffusion of protein molecules and allow the formation of crystals suitable for crystallographic studies.9
  10. Growth of protein crystals

Properties of Agarose

Please refer to the table below.

Agarose is non-toxic and can be handled freely.7

Sulfate content may be used as an indicator of purity, since sulfate is the major ionic group present. Lower sulfate content is preferable for DNA electrophoresis.

Gel strength is the force that must be applied to a gel to cause it to fracture. Gel strength is dependent on the concentration of the gel and therefore the gel strength is always reported with a specific percent agarose. Agarose is unique in that it can produce strong gels with a low concentration of agarose.

The gel point is the temperature at which an aqueous agarose solution forms a gel as it cools. Agarose solutions exhibit hysteresis in the liquid-to-gel transition - that is, the gel point of the solution is NOT the same as its melting temperature. In general, the gelling temperature range, also known as the transition temperature is 32-45 °C and the melting temperature range is 80-95 °C.

Anionic groups in an agarose gel are affixed to the matrix and cannot move, but dissociable cations can migrate toward the cathode in the electrophoresis unit, giving rise to electroendosmosis (EEO) - a movement of liquid through the gel. Since electrophoretic movement of biopolymers is usually toward the anode, EEO can disrupt separations because of internal convection.(6, 10)

Usage Instructions for making Gels

Boiling water bath method:

  1. Add any buffer of choice (usually with an ionic strength, µ,2 of 0.03-0.10) and a stir bar to a beaker which can hold 2-4 times the volume of the desired solution.3
  2. Slowly sprinkle the agarose powder into the liquid while stirring to prevent clumping.
  3. Weigh the beaker and solution before heating.
  4. Cover the beaker with plastic wrap and pierce a hole in the wrap for ventilation.
  5. Bring the solution to a boil and allow it to boil for 5-10 minutes stirring continuously, until agarose dissolves completely. To avoid charring, use a boiling water bath rather than directly applied heat.3
  6. Add enough hot distilled water to return the contents to the original weight; mix continuously.
  7. Allow the mixture to cool to 50-55 °C, at which temperature it is ready to be cast into cassettes which have been pre-warmed to 50-55 °C.3

Microwave method 1 (for gels ≤2% w/v):

  1. Add any buffer of choice, usually with an ionic strength, µ of 0.03-0.10 (μ = 2 Si Ci x Zi2, where Ci = molar concentration of a given ion Zi = charge of a given ion)2 and a stir bar to a beaker which can hold 2-4 times the volume of the desired solution.3
  2. Slowly sprinkle the agarose powder into the liquid while stirring to prevent clumping.
  3. Remove the stir bar.
  4. Weigh the beaker and solution before heating.
  5. Cover the beaker with plastic wrap and pierce a hole in the wrap for ventilation.
  6. Place the solution in a microwave oven and heat on HIGH power for 2 minutes.
  7. Remove the solution from the microwave oven very carefully; any microwaved solution may be superheated and could foam over the container's rim if agitated. Swirl gently to re-suspend any remaining agarose particles.
  8. Reheat on HIGH power for 1-2 minutes or until the solution comes to a boil. Boil for 1 more minute or until the solution is clear and the agarose is completely dissolved.
  9. Remove the solution from the oven very carefully and swirl it gently.
  10. Add enough hot distilled water to return the contents to the original weight; mix continuously.
  11. Allow the mixture to cool to 50-55 °C, at which temperature it is ready to be cast into cassettes which have been pre-warmed to 50-55 °C.3

Microwave method 2 (for gels >2% w/v):

Follow the same protocol as for ≤2% gels (above), but use a MEDIUM instead of HIGH power setting in step 6.

Other Gelling Agents

  1. Agar: Separate data sheet is available.

  2. Gelatin: Separate data sheet is available.

  3. Phytagel (Product No. P8169):Phytagel is an agar substitute produced from a bacterial carbohydrate composed of glucuronic acid, rhamnose, and glucose. It produces a clear, colorless, high strength gel which aids in detection of microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent (agar contains about 70% agarose). Originally developed for microbial applications, Phytagel is a good choice in any application (such as plant cell culture) where some agar products tend to inhibit growth due to unidentified impurities. To prevent clumping, Phytagel should be added to culture medium that is at room temperature, with rapid stirring.

  4. Agargel (Product No. A3301):Agargel is a blend of agar and Phytagel which was developed to help control vitrification (also known as hyperhydricity which is a physiological abnormality of tissue culture-generated plants) in plant tissue cultures. Agargel provides the positive attributes of agar and Phytagel, is cheaper than agar, and is superior to Phytagel in applications where vitrification is a problem. Agargel produces a semiclear gel which allows for better detection of contamination.

 References

  1. Gel Filtration Principles and Methods, 5th Ed., Pharmacia LKB Biotechnology (1991).
  2. T. G. Cooper, The Tools of Biochemistry, p. 176, John Wiley & Sons, New York (1977).
  3. A. T. Andrews, Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications, 2nd Ed., p. 149, A. R. Peacock and W. F. Harringdon, Eds., Oxford Science Publications, Clarendon Press, Oxford (1993).
  4. Araki, Choji. "Structure of the agarose constituent of agar-agar." Bulletin of the Chemical Society of Japan 29.4 (1956): 543-544.
  5. Viljoen, C. D., B. D. Wingfield, and M. J. Wingfield. "Agar, an alternative to agarose in analytical gel electrophoresis." Biotechnology techniques 7.10 (1993): 723-726.
  6. Serwer, Philip. "Agarose gels: properties and use for electrophoresis." Electrophoresis 4.6 (1983): 375-382.
  7. Olsson, Ingmar, and Torgny Låås. "Isoelectric focusing in agarose under denaturating conditions." Journal of Chromatography A 215 (1981): 373-378.
  8. Crowle, Alfred J. "Immunodiffusion." Methods of Protein Separation. Springer, Boston, MA, 1975. 69-92.
  9. Otálora, Fermín, et al. "Counterdiffusion methods applied to protein crystallization." Progress in biophysics and molecular biology 101.1-3 (2009): 26-37.
  10. Guo, Yaojun, Xinhui Li, and Yong Fang. "The effects of electroendosmosis in agarose electrophoresis." Electrophoresis 19.8‐9 (1998): 1311-1313.

 

Agarose Product Overview

Product No. Description Sulfate Gel Strength
(g/cm2)
Gelling Temp.
(°C)
Melting Temp.
(°C)
EEO
A6013 Agarose Type I: Low EEO ≤0.15% ≥1200 at 1.0% 36±1.5 at 1.5%
Gel Point
N/A 0.09-0.13
A0169 Agarose Type I-A: Low EEO ≤0.2% ≥1200 at 1.0% 36±1.5 at 1.5%
Gel Point
88±1.5 0.09-0.13
A0576 Agarose Type I-B: Low EEO
  • High gel strength
  • Suitable for separating high molecular weight
≤0.12% ≥3200 at 1.5% 36±1.5 at 1.5%
Gel Temp.
88±1.5 ≤0.12
A9539 Agarose: Low EEO, For Routine Use
Molecular Biology Reagent

  • DNase and RNase: none detected.
  • For analysis and purification of nucleic acids
≤0.15% ≥1200 at 1.0% 36±1.5 at 1.5%
Gel Point
N/A 0.09-0.13
A4679 Agarose: Low EEO, For
Immunoelectrophoresis
Electrophoresis Reagent

Suitable for standard immunoelectrophoresis and immunodiffusion
≤0.20% ≥1200 at 1.0% 36±2.0 at 1.5%
Gel Point
88±2.0 at 1.5% 0.09-0.13
AGRLE-RO Agarose: Low EEO
For use in standard gel electrophoresis
≤0.14% ≥1200 at 1.0% ≥2500 at 1.5% 36±1.5 at 1.5% Gel Temp. 88±1.5 at 1.5% 0.05-0.13
A6877 Agarose Type II: Medium EEO ≤0.20% ≥1000 at 1.0% 36±1.5 at 1.5% Congealing Temp. N/A 0.16-0.19
A9918 Agarose Type II-A: Medium EEO ≤0.25% ≥1000 at 1.0% 36±1.5 at 1.5%
Gel Point
88±1.5 0.16-0.19
A6138 Agarose Type III: High EEO ≤0.20% ≥650 at 1.0% 36±1.5 at 1.5%
Gel Temp.
N/A 0.23-0.26
A9793 Agarose Type III-A: High EEO ≤0.25% ≥750 at 1.0% 36±1.5 at 1.5%
Gel Temp.
N/A 0.23-0.26
05066 Agarose: High EEO
For Molecular Biology

DNase, RNase, Phosphatases, Proteases: none detected.
≤0.6% ≥1500 34-37 at 1.5%
Gel Temp.
N/A 0.23-0.27
A3643 Agarose Type IV: Special High EEO
  • Lower sulfate content and non-specific protein binding capacity than Type III
  • For electrophoretic techniques requiring a high degree of cathodal movement
N/A N/A N/A N/A ≥0.30
A5304 Agarose: Special High EEO
Electrophoresis Reagent
Suitable for counterimmunoelectrophoresis and immunoelectrophoretic techniques with significant cathodal migration.
≤0.2% ≥700 at 1.0% 36±1.5 at 1.5%
Gel Point
N/A ≤0.40
A7174 Agarose Type VI-A: High Gelling Temp. ≤0.20% ≥900 at 1.0% 41±1.5 at 1.5%
Gel Point
N/A ≤0.14
A0701 Agarose Type VII-A: Low Gelling Temp. Excellent for in-gel enzymatic reactions and cloning assays and for recovery of heat-labile samples after electrophoresis. ≤0.4% ≥250 at 1.0% 26±2.0 at 1.5%
Gel Point
≤65.5 ≤0.12
A4018 Agarose: Low Gelling Temp.
  • A low gelling temperature derivative
  • Gels form at <30 °C and remelts at temperatures >65 °C
  • Useful for media containing heat-labile materials
N/A ≥200 at 1.0% 26-30 Congealing Temp. N/A ≤0.10
A9414 Agarose: Low Gelling Temp.,
For Molecular Biology
Molecular Biology Reagent

DNase, RNase and NICKase: none detected.
≤0.10% ≥200 at 1.0% 26-30 Congealing Temp. ≤65 ≤0.10
A4905 Agarose Type VIII
For isoelectric focusing.
≤0.2% ≥500 at 1.0% N/A N/A ≤0.02
A5030 Agarose Type IX: Ultra-low Gelling Temp. Gelling occurs at 8-17 °C and remelting at <50 °C N/A N/A 8-17 at 0.8%
Gel Temp.
≤50 ≤0.05
A2576 Agarose Type IX-A: Ultra-low Gelling Temp.
  • Strong gels with ultra-low gelling temperature
  • For electrophoresis of heat-labile samples
  • For growth of hybridomas and other cell lines
≤0.14% ≥300 at 1.5% ≤20 at 1.5%
Gel Point
≤62 at 1.5% ≤0.12
A3038 Agarose Type XI: Low Gelling Temp.
Suitable for separation of small nucleic acid fragments.
≤0.15%

N/A

≤35
Gel Temp.
N/A ≤0.15
A7299 Agarose Type XII: Low Viscosity for Beading Recommended for preparation of agarose beads. ≤0.20% ≥900 at 1.0% 41±1.5 at 1.5%
Gel Temp.
N/A ≤0.14
A2929 Agarose: For Pulsed Field Electrophoresis
Running Gel

Molecular Biology Reagent
  • DNase, RNase, and NICKase: none detected
  • Suitable for the separation of high molecular weight DNA
≤0.20% ≥2000 38-43 at 10%
Gel Temp.
N/A ≤0.10
A3054 Agarose: For Pulsed Field Electrophoresis Sample Preparation
Molecular Biology Reagent
  • DNase and RNase: none detected
  • Suitable for making gel plugs when separating high molecular weight DNA without the shearing encountered by conventional isolation techniques
≤0.15% N/A 26-30
Gel Temp.
N/A ≤0.10
A2790 Agarose: Wide Range
Molecular Biology Reagent
  • DNase and RNase: none detected
  • Capable of separating DNA fragments with 50-1,000 base pairs on a single 3% gel
<0.15% N/A ≤35
Gel Temp.
≤65 ≤0.15
A7431 Agarose: Wide Range/Standard 3:1 Ratio, For Molecular Biology
Molecular Biology Reagent
  • DNase, RNase, and NICKase: none detected
  • Composed of 3 parts low gelling temperature agarose and 1 part high gelling temperature agarose
  • Specially formulated to form strong, flexible gels for separation of small (#1 kb) PCR products, DNA and RNA
≤0.15% ≥1200 at 4.0% 32.5-38 at 4%
Gel Temp.
≤90 at 4% ≤0.13
A4718 Agarose: High Resolution, for Molecular Biology
DNase, RNase, and NICKase: none detected.
N/A N/A ≤35 at 3%
Gel Temp.
≤75 at 3% ≤0.05
AGRMP-RO Agarose: Multipurpose
DNase, RNase: none detected.
≤0.12% ≥1800 at 1.0% ≥3200 at 1.5% 36±1.5 at 1.5%
Gel Temp.
88±1.5 at 1.5% ≤0.12

N/A = Not Available
N/D = Not Detectable
— = Not Applicable