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Native Agarose Gel Electrophoresis of Multiprotein Complexes

Rosalind Kim

Cold Spring Harb Protoc 2011; doi: 10.1101/pdb.prot4558

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Characterization of Protein Complexes (58 articles)
Electrophoresis of Proteins (76 articles)
Protein Classification and Structure Prediction (158 articles)
Protein Identification and Analysis (158 articles)
Protein: Protein Interactions (50 articles)
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Protocol

Native Agarose Gel Electrophoresis of Multiprotein Complexes

Rosalind Kim

INTRODUCTION
An important tool for the biochemist is the ability to analyze proteins in their native state. Electrophoresis
of proteins and protein–protein complexes in native agarose gels using a horizontal gel apparatus is
described here. The procedure is simple to set up, takes a short time to run, and avoids the use of
toxic components. The gel is run in a submerged horizontal platform, with the wells positioned in the
center of the gel. Proteins with a pI lower than the buffer pH carry a net negative charge and migrate
toward the anode, whereas proteins with a pI higher than the buffer pH carry a positive charge and
migrate toward the cathode. Proteins with different molecular weights and pI values can be tested,
as can proteins that form complexes, whether they be two pure proteins forming a complex or a
complex formed after incubating a pure protein with a crude extract. Once a complex is formed, it
can be cut from the gel and the components can be isolated. This method does not replace isoelectric
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focusing (IEF) gels because it does not determine the pI of a protein, but it does facilitate the detection
of protein–protein complexes and may give information on the protein’s charge at a defined pH.

RELATED INFORMATION
Protocols are available for preparation of Yeast Protein Extracts (Amberg et al. 2006), SDS-PAGE of
Proteins (Simpson 2006), Extraction and Solubilization of Total Protein from Microorganisms
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(Görg et al. 2006a), Extraction and Solubilization of Total Protein from Plant Seeds (Görg et al.
2006b), Extraction and Solubilization of Total Protein from Mammalian Tissue Samples (Görg
et al. 2006c), Preparation and Use of an Integrated Microcapillary HPLC Column and ESI Device
for Proteomic Analysis (Goodlet et al. 2007), and a General Method for MALDI-MS Analysis of Pro-
teins and Peptides (Frecklington 2007).

MATERIALS
PROTOCOLS

RECIPES: Please see the end of this article for recipes indicated by <R>.
It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s
Environmental Health and Safety Office for proper handling of equipment and hazardous materials
used in this protocol.

Reagents

<R> 2× Sample-loading buffer

Agarose (powdered)
Cellular extract containing proteins of interest (optional; see Step 2)
For the preparation of suitable extracts, see Yeast Protein Extracts (Amberg et al. 2006), Extraction and Solu-
bilization of Total Protein from Microorganisms (Görg et al. 2006), Extraction and Solubilization of Total
Protein from Plant Seeds (Görg et al. 2006), and Extraction and Solubilization of Total Protein from Mamma-
lian Tissue Samples (Görg et al. 2006).
<R> Gel destaining solution

Adapted from Proteins and Proteomics (ed. Simpson).

CSHL Press, Cold Spring Harbor, NY, USA, 2003.
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<R> Gel staining solution

Glycerol (5%)
<R> Native gel buffer (Buffer A)
Pure protein samples for analysis (2–5 µg per lane) (see Step 2)
SDS–polyacrylamide gel
Prepare an SDS–polyacrylamide gel according to SDS-PAGE of Proteins (Simpson 2006) or use a precast gel (e.g.,
Amersham Biosciences/GE Healthcare) (see Step 15).

Equipment
Beaker
Cellophane (ultraclear) (Idea Scientific Co.)
This cellophane is used for drying the agarose or polyacrylamide gels. Even though the agarose gels can be as thick
as 0.7 cm, these gels (after soaking in 5% glycerol) can be placed between two layers of cellophane, stretched
using a frame, and dried for 24 h at 37˚C to preserve the data.
Centrifuge
Erlenmeyer flask (see Step 1 for size needed)
Gel-casting assembly
Gel electrophoresis apparatus (horizontal) (e.g., Horizon 58; Biometra)
Microcon centrifugal filter units (Millipore)
Micropipettor and tips
Microwave oven
Oven preset to 37˚C
Razor blade
Scale
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Ultrafree-DA centrifugal filter unit (Millipore) (see Step 11)

Water bath

METHOD
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Preparation of the Native Gel

1. Prepare a horizontal 0.8% agarose gel in buffer A. The dimensions of the gel should be
8 cm ×
5.5 cm × 3 mm.
i. Add powdered agarose and buffer A to an Erlenmeyer flask that is three to four times the
volume of buffer A used. Weigh the flask.

ii. Cover the flask with an inverted beaker and place it in a microwave until the agarose melts.

iii. Reweigh the flask and add H2O, if necessary, to bring it back to its original weight.
PROTOCOLS

iv. Allow the agarose to cool to 45˚C in a water bath.

v. Place the comb in the center of the gel-casting assembly and pour the agarose solution into
the assembly.

vi. Allow the agarose to harden.

vii. Place the gel into a horizontal electrophoresis unit containing buffer A. Make sure that the
agarose gel is completely submerged. Carefully remove the comb.

Identification of Protein Complexes

2. Using purified proteins, incubate the proteins of interest together under conditions that facilitate
protein complex formation. To determine whether proteins in a cell extract may bind to a protein
of interest, incubate 5 µg of the pure protein with 45 µg of soluble cell extract under the appropriate
conditions for forming protein complexes.
3. Mix 2–5 µg of each protein sample 1:1 (v/v) with 2× sample-loading buffer.
The protein samples should include each individual protein (and cell extract, if used) and the mixtures incubated as in
Step 2.

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FIGURE 1. Schematic representation of migration of negatively

and positively charged proteins and a complex of the two.
Native agarose gel (0.8%) is shown as described in this protocol.
(Lane 1) A positively charged protein; (lane 3) a negatively
charged protein; (lane 5) a complex of the two proteins (lanes
2,4 are blank).

4. Load the samples into the wells (typically, load
20 µL per well). Run the gel at a constant voltage of
50 V for 1 h at room temperature.
5. Stain the gel in gel-staining solution for 20 min. Destain in gel-destaining solution until the protein
bands can be clearly identified (see Fig. 1).
6. To dry the gel, soak it in 5% glycerol for 5 min. Dry it between two sheets of cellophane membrane
for 24 h at 37˚C.
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Isolation and Identification of the Components of a Protein–Protein Complex

7. Load two identical protein complex samples into adjacent lanes of a 0.8% agarose gel. Run the gel at
a constant voltage of 50 V for 1 h at room temperature.
8. Use a sharp razor blade to separate the two lanes from the gel.
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9. Stain one of the two lanes as described in Step 5. Keep the other lane submerged in buffer A.

10. Identify the region of the stained gel that contains the protein complex to be recovered. Using the
stained gel as a guide, excise the slice of agarose from the unstained gel fragment that corresponds to
the complex.
11. Place the unstained gel fragment containing the complex into an Ultrafree-DA unit. Place the
filter unit into a filtrate vial. Centrifuge the unit at 5000g for 10 min at either room temperature
or 4˚C.
The gel nebulizer in the Ultrafree-DA unit is a device that will convert the gel slice to a fine spray upon centrifugation.
PROTOCOLS

The gel fragment must pass through the orifice of the nebulizer, thus converting the gel to a fine slurry. The gel particles
are captured by the filter unit and discarded (the filter unit is not reusable).

12. Transfer the protein sample in the filtrate vial to a Microcon centrifugal filter unit (the cutoff size to
use is determined by the size of the proteins under study).
13. Centrifuge the Microcon concentrator at 14,000g until the volume of the sample is reduced to
10–20 µL.
The proteins are retained above the membrane in the Microcon sample reservoir.

14. Recover the concentrated proteins by inverting the Microcon into a new vial. Centrifuge at 1000g
for 3 min.
For complete details, refer to the manufacturer’s instructions.

15. To identify the sizes of the individual components of the protein complex band, analyze the concen-
trated proteins by SDS-PAGE (see SDS-PAGE of Proteins) (Simpson 2006).
The percentage of acrylamide in the gel will depend on the sizes of the proteins under study. Alternatively, the proteins
(
4 pmoles are required) can be analyzed by electrospray ionization (ESI) or matrix-assisted laser desorption/ioniz-
ation (MALDI)- mass spectrometry (MS) (see, e.g., Preparation and Use of an Integrated Microcapillary HPLC
Column and ESI Device for Proteomic Analysis [Goodlet et al. 2007], and a General Method for MALDI-MS Analysis
of Proteins and Peptides [Frecklington 2007]).

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REFERENCES
Amberg D, Burke DJ, Strathern JN. 2006. Yeast protein extracts. Cold
Spring Harb Protoc doi: 10.1101/pdb.prot4152.
Frecklington D. 2007. General method for MALDI-MS analysis of pro-
teins and peptides. Cold Spring Harb Protoc doi: 10.1101/pdb.
prot4679.
Goodlet DR, Yi EC, Mottay P. 2007. Preparation and use of an inte-
grated microcapillary HPLC column and ESI device for proteomic
analysis. Cold Spring Harb Protoc doi: 10.1101/pdb.prot4617.
Görg A, Drews O, Weiss W. 2006a. Extraction and solubilization of
total protein from microorganisms. Cold Spring Harb Protoc doi:
10.1101/pdb.prot4224.
Görg A, Drews O, Weiss W. 2006b. Extraction and solubilization of total
protein from plant seeds. Cold Spring Harb Protoc doi: 10.1101/
pdb.prot4225.
Görg A, Drews O, Weiss W. 2006c. Extraction and solubilization of total
protein from mammalian tissue samples. Cold Spring Harb Protoc
doi: 10.1101/pdb.prot4226.
Simpson RJ. 2006. SDS-PAGE of proteins. Cold Spring Harb Protoc doi:
10.1101/pdb.prot4313.

RECIPES
Recipes for items marked with <R> are provided here. Additional recipes can be found online at http://
www.cshprotocols.org/recipes.

2X Sample-loading buffer
20% glycerol
0.2% bromophenol blue
<R> 0.12 M Tris-Cl
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Gel destaining solution

45% methanol
10% acetic acid
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Gel staining solution

0.12% Coomassie Brilliant Blue R
45% methanol
10% acetic acid

Native gel buffer (Buffer A)

25 mM Tris-Cl (pH 8.5)
19.2 mM glycine
PROTOCOLS

Tris-Cl
Tris base
HCl
To prepare a 1 M solution, dissolve 121.1 g of Tris base in 800 mL of H2O. Adjust the pH to the desired value by
adding concentrated HCl.

PH HCl
7.4 70 mL
7.6 60 mL
8.0 42 mL
Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the
solution to 1 L with H2O. Dispense into aliquots and sterilize by autoclaving.
If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is tempera-
ture-dependent and decreases
0.03 pH units for each 1˚C increase in temperature. For example, a 0.05 M solution
has pH values of 9.5, 8.9, and 8.6 at 5˚C, 25˚C, and 37˚C, respectively.

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