Protein Separationin Agarose Gels

Section XIII: Protein Separation in Agarose Gels

In This Section
Introduction
196
Buffers for Protein Separation in Agarose
197
Casting Agarose Gels for Protein Separation
198
Preparation and Loading of Protein Samples
198
Optimal Voltage and Electrophoretic Times
199
Detection of Proteins in Agarose Gels
199
Gel Drying and Preservation
200
Processing Agarose Gels Following Electrophoresis 200
Recovering Proteins from Agarose Gels
201
References
202
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Section XIII: Protein Separation in Agarose Gels
Introduction

Protein electrophoresis in agarose gels is an alternative
approach to using polyacrylamide gels and provides
several benefits. Gels can be run using a vertical systemor a horizontal system and unlike polyacrylamide gels,
agarose gels can be used effectively to separate proteinslarger than 600,000 Da.
Advantages

Recommended agaroses for protein electrophoresis
The table below is a list of Lonza Agaroses that arerecommended for protein electrophoresis. For performing
routine separations, we recommend a standard melting
temperature agarose such as MetaPhor® Agarose orSeaKem® Gold Agarose. When proteins are to be recovered
for further analysis, use a low melting temperature agarosesuch as SeaPlaque® GTG® or NuSieve® GTG® Agarose.

—— Separate high-molecular-weight proteins(>600,000 Da)—— Efficient recovery of proteinsProtein Size
Range(kDa)
20 - 200—— Excised proteins can be used to immunize animals directly
for antibody production

150 - 300—— Easy to prepare and handle—— Non-toxic

300 - 600
600 - 1,000
1,000 - 5,000

AgaroseConcentration

MetaPhor® orNuSieve® GTG®
MetaPhor® orNuSieve® GTG®
MetaPhor® or
NuSieve® GTG®
SeaKem® Gold or
SeaPlaque®
SeaKem® Gold or
SeaPlaque®

5%
3%
2%
1.5%
1.0%

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Section XIII: Protein Separation in Agarose Gels
Buffers for Protein Separation in Agarose

The buffer systems used for agarose electrophoresis aresimilar to those used for polyacrylamide electrophoresis.
When performing horizontal electrophoresis, we havefound that a Tris-borate gel and running buffer provide
greater resolution than using the standard Laemmli Buffersystem.
Vertical and horizontal gel
1X Stack Buffer, pH 8.0
125 mM Tris-HCl

g/l for 1X Stack Buffer
19.7 g Tris-HCl in 1 liter 		distilled water
Note: Horizontal gels do not require the use of a stack gel.
1X Resolving Buffer, pH 8.5 g/l for 1X Resolving Buffer
500 mM Tris base60.55 g Tris base160 mM Boric acid9.90 g Boric acid
1 M Urea
60.06 g Urea
Adjust volume to 1 liter with distilled water
1X Running Buffer, pH 8.5 amount for 1X RunningBuffer
90 mM Tris base10.90 g Tris base
90 mM Boric acid
5.57 g Boric acid
0.1% SDS
10 ml of 10% SDS
Adjust volume to 1 liter with distilled water

Laemmli Buffer—— Gels can be made with standard Laemmli Buffer (seepage 156).
—— To avoid excess foaming during agarose dissolution; only
add SDS to the cathodal buffer and not to the gel buffer.
The SDS in the cathodal buffer will migrate faster thanthe proteins during electrophoresis, maintaining protein
denaturation.
—— To prevent buffer depletion in vertical systems, use
10X Laemmli Buffer without SDS in the anodal buffer.
Tips for buffer preparation—— Add SDS to the cathodal buffer only.
—— Do not add SDS to the gel prior to dissolution.
—— If other buffer systems are used, the pH should bebetween pH 5-9.
—— Denaturants such as urea and formamide should only
be added at low concentrations (4 M-6 M Urea).
—— For buffers more alkaline than pH 9, dissolve andcast the agarose in distilled water, allow to gel. Soakhorizontal gels in the alkaline gel buffer for 30 minutes
prior to electrophoresis. For vertical gel systems,
prerun the gel to equilibrate the gel with the alkaline
buffer.
Caution: Materials and methods shown here present
hazards to the user and the environment. Refer tothe safety information on page 210 before beginningthese procedures.

Protein Separationin Agarose Gels

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Section XIII: Protein Separation in Agarose Gels
Casting Agarose Gels for Protein Separation

The procedures for dissolving and casting agarose gels forprotein separation are the same as the procedures used
for nucleic acid separation. Refer to Dissolving Agaroseand Casting Agarose Gels (see Section II).
Tips for casting horizontal agarose gels—— Use of a stacking gel is not necessary for horizontal
submarine electrophoresis.
—— The resolving gel buffer and running buffer should bethe same.
–– Dissolve the agarose in running buffer without
SDS.
–– 	For denaturing electrophoresis, add SDS to thesample buffer and the running buffer.
—— Let the gel set for 20-30 minutes at room
temperature.
—— For MetaPhor® and SeaPlaque® Agarose, chill the gelat 4°C for 20-30 minutes before removing comb.
—— If gels are to be dried, cast the gels onto GelBond®
Film.
Tips for casting vertical agarose gels■■ Stacking gel for vertical gels—— Flood the comb area with running buffer prior toremoving the comb.
—— If clamps are used, remove the clamps at the top of thegel cassette and gently loosen the comb by moving it
forward and back before removal.
Preparation and Loading of Protein Samples
Sample preparation and amount of protein that can beloaded on agarose gels is essentially the same as forpolyacrylamide gels and is largely dependent on yourapplication and detection method.
Guidelines—— Suspend protein samples in 2X sample buffer, 1:1(v:v).
—— If denatured proteins are required, incubate at
95°C-100°C for 5 minutes.
—— Load the samples into the sample wells.
—— The minimal amount of protein detectable byCoomassie® Brilliant Blue stain is about 1.0 µg; andmay vary depending on the protein.
—— Larger amounts of protein can be loaded, but band
thickness increases accordingly.

—— For proteins >100 kDa, the use of a stacking gel may
be omitted. It will not enhance band resolution.

—— For a 0.8 cm wide well, 25 ml (50 µg total protein) is
recommended for a complex mixture, if staining withCoomassie® Blue, and 1 ml (10 µg total protein) isneeded for samples containing one or a few proteins.

—— After the stacking gel is set, place cassette at 4°C for
30 minutes prior to removing the comb.

—— For vertical electrophoresis, load empty wells withsample buffer.

—— Prepare a 1% SeaKem® Gold Agarose gel in stacking
gel buffer.

Protein Separationin Agarose Gels■■ Resolving gel for vertical gels—— Dissolve the agarose in running buffer without SDS.
—— Refer to Vertical Gel Casting Instructions (see page86).
—— Allow resolving gel to set approximately 3 minutes atroom temperature then cast the stacking gel.
■■ To facilitate comb removal from a vertical gel—— The teeth of the comb can be tapered so the width at
the bottom is slightly smaller than at the top. A slightrounding of the edges is all that is needed so that theend is U-shaped. Tapering the teeth in this way will notaffect the pattern of the protein bands.

2X Tris-Glycine SDS sample buffer for agarose
electrophoresis of proteins
2X concentrate
126 mM Tris-HCl, pH 6.8

Amount to add for 10 ml
2.5 ml of 0.5 M Tris-HCl, 		pH 6.8®
15% Ficoll Type 400
1.5 g Ficoll Type 400
4% SDS
4 ml of 10% SDS
0.002% Bromophenol Blue 0.2 ml of 0.1% 			Bromophenol blue
Adjust volume to 10 ml with distilled water
Before use: add 1 ml β-Mercaptoethanol (βME) to 10 ml of2X Tris-Glycine SDS sample buffer

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Section XIII: Protein Separation in Agarose Gels
Optimal Voltage and Electrophoretic Times■■ Tips—— Avoid higher power settings as the heat generated
may melt the agarose.
—— Thick vertical gels (>1 mm) will require proportionallyhigher current settings to complete the electrophoresis
run within the times indicated.

—— Prestained molecular weight markers such as Lonza’sProSieve® Color Protein Marker can be used to monitor
electrophoresis. The gels can be electrophoresed
longer, but care should be taken that smaller proteins
do not travel off the gel.

—— Electrophorese the gel until the tracking dye travels tothe bottom of the resolving gel.
Horizontal Gel
Gel Size 5.3 cm x 8.5 cm x .4 cm
Power Setting 100 volts				Time 3 - 4 hours
Gel Type

Vertical Gel
14.5 cm x 16.5 cm x .1 cm
25 mA (constant)(approximately 80 volts at thestart and 200 volts at the end)
3 - 3.5 hours

Mini-Vertical
8 cm x 10 cm x .1 cm20mA(approximately 50 volts at thestart and 120 volts at the end)
1.5 - 2 hours

Section XIV: Protein Separation in Agarose Gels
Detection of Proteins in Agarose Gels
The procedures for staining agarose gels with Coomassie®
Blue Stain are essentially the same as they are forpolyacrylamide gels with some modifications (listedbelow). For detailed procedures on Coomassie® Brilliant
Blue staining (see page 168), using the modifications
listed below. For detailed procedures on Silver staining,refer to Staining Proteins with Silver Stain (see page 186
in Isoelectric Focusing of Proteins on Agarose Gels).
Tips—— Agarose gels require more time to process thanpolyacrylamide gels of similar dimensions.
—— Staining and destaining times will vary dependingon the gel concentration, thickness and protein
concentration.
—— Place container on shaker with gentle motion during
staining and destaining procedures.
Staining proteins with Coomassie® Brilliant Blue StainCoomassie® Blue Stain solution Destain Solution
40% Methanol
20% Methanol
10% Glacial Acetic Acid
5% Glacial Acetic Acid
0.25% Coomassie® Brilliant Blue R-250

Room temperature staining—— A 14.5 cm x 16.5 cm, 1 mm thick agarose gel will stain
in approximately 1 to 2 hours.
—— Destain for 1-4 hours with gentle shaking at room
temperature.
Overnight staining

Protein Separationin Agarose Gels

Returnto Main—— Use 0.125% Coomassie® Blue R-250 with the same
concentrations of methanol and acetic acid as in thestain solution.
—— Destain approximately 4 hours.
Accelerated staining—— Stain gels using standard stain solutions at 50°C.
—— A 1 mm thick gel takes approximately 1 hour to stain
and 1 hour to destain.
—— Change the destaining solution 1 time.
—— Agarose gels become softer at 50°C - use a support totransfer between solutions.
Storage—— Do not store agarose gels in destain solution, they
may become brittle and fracture.
—— Store gels in a 5% glycerol solution or dried.
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Returnto MainSection XIII: Proteins Separation in Agarose Gels
Gel Drying and Preservation

Agarose gels can be dried overnight at room temperature,dried in a forced hot-air oven or dried using a standard
vacuum gel dryer. When not using a vacuum gel dryer,
the gel must first have been cast onto GelBond® Film toprevent the gel from shrinking during the drying process.
The procedures for drying protein agarose gels are thesame as drying DNA agarose gels.

Proteins Separation in Agarose Gels
Processing Agarose Gels Following Electrophoresis

Autoradiography

Electroblotting proteins from agarose gels

After drying, agarose gels can be exposed directly to X-rayfilm.

Proteins can be electroblotted out of agarose gels onto
membranes (nitrocellulose, PVDF, etc.) by using the same
methods used for polyacrylamide gels. Refer to Blotting
Proteins from Polyacrylamide Gels for detailed procedures(see page 171). It is important to note that agarosegels adhered to GelBond® Film cannot be electroblottedbecause GelBond® Film is nonporous. The time requiredfor optimal transfer of specific proteins will need to be
determined experimentally. In general, proteins transfer
15% faster out of agarose gels than from a polyacrylamidegel.

Fluorography

Protein Separationin Agarose Gels

Do not immerse agarose gels into any fluorography
solution if the gels are attached to GelBond® Film. Solutions
containing high concentrations (>50%) of DMSO must not
be used, as they will dissolve the agarose. Commerciallyprepared solutions which precipitate the fluor within theagarose gel matrix (e.g., EN3HANCE® from Perkin Elmer)
work best. Follow the manufacturer’s instructions. The
fluor-impregnated gel can then be dried onto filter paper
under vacuum at <50°C in a slab-gel dryer and thenexposed directly to X-ray film.

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Section XIII: Proteins Separation in Agarose Gels
Recovering Proteins from Agarose Gels

Proteins can be readily recovered from agarose gels.
When protein is to be recovered, the use of a low melting
temperature agarose such as NuSieve® GTG,® MetaPhor® orSeaPlaque® Agarose is recommended.

■■ Materials–– Spatula–– 1.5 ml microfuge tube(s)–– Heat block

Protein Recovery Tips–– -70°C freezer—— Identifying the protein to be recovered from the gel
can be accomplished by several methods. If thegel is fixed and stained with Coomassie® Blue asdetailed earlier, then 1% SDS should be added to thedilution buffer. Recovery of proteins in the native
state requires that they not be fixed prior to recovery.
Methods for detecting proteins which do not require
fixing include:–– Microcentrifuge at 4°C■■ Reagents–– Extraction buffer (50 mM Tris-HCl, 1 mM EDTA at pH8.0)–– Ice–– 	Using prestained molecular weight markers asa guide for the relative position of the desired
protein in the gel.
–– Performing a short (<10 minutes) pressure/capillary blot of the gel so that small (<5%)
amounts of the proteins are transferred
to a membrane and then gold stained. If
prestained molecular weight markers are used, it
is possible to place the stained membrane under
the gel and identify the region of interest.
—— We recommend the use of 50 mM Tris-HCl, 1 mM EDTA at
pH 8.0 as the extraction/dilution buffer. The buffer may
require modification depending upon the particularprotein to be recovered and what further work isplanned after it is recovered. The buffer should be one
in which the protein of interest will be stable.

Protein Separationin Agarose Gels

Returnto Main—— The amount of extraction/dilution buffer added is
directly related to the amount of protein that can berecovered: the more the agarose is diluted, the more
protein will be recovered. Greater than 90% of a protein
can be recovered with a dilution to 0.5% agarose. A
second dilute-freeze-spin cycle can be performed torecover additional protein.

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Section XIII: Proteins Separation in Agarose Gels
Recovering Proteins from Agarose Gels — continued

Procedure
1.

Identify the region of the gel which contains the protein
to be recovered.

2.

Excise a gel slice containing the protein of interest.

3.

Place the gel slice in a 1.5 ml microfuge tube.

4.

Determine the volume of the gel slice by weight or size.

5.

Add the appropriate amount of extraction buffer so thefinal concentration of agarose is 0.5%.

6.

Melt the gel slice by heating to 70°C for SeaPlaque® orNuSieve® GTG® Agarose or 80°C for MetaPhor® Agarose.

7.

Mix thoroughly.

Note: Ensure the gel is completely melted and diluted by thebuffer.
8.

Incubate on ice for 30 minutes.

Note: It is important that the agarose mixture has gelled asmuch as possible at this step.
9.

Freeze the mixture at -70°C for 1 to 2 hours.

10.

Allow the mixture to thaw on ice.

11.

Centrifuge the mixture at 13,000 rpm in a

microcentrifuge for 10 to 20 minutes at 4°C.
12.

Remove the supernatant; this contains the recovered
protein.

Protein Separationin Agarose Gels

Note: Each step of this procedure should be carefully followed toobtain quantitative results. Care should be taken to COMPLETELY
gel the protein-agarose solution. If the procedure cannot becompleted at one time, we recommend keeping the mixture
at -70°C, as prolonged freezing will not affect the protein.
Protein recovered from MetaPhor® Agarose by this techniquewill co-purify with some residual agarose. The amountof agarose present is between 1% and 3% of the originalamount of agarose present in the gel slice. All of the residualagarose will pass through a 0.1 mm pore filter, about 83%
will pass through a 100 kDa molecular-weight cutoff filter, and50% will pass through a 30 kDa molecular-weight cutoff filter.
The recovered protein can be separated from the residual
agarose by conventional chromatography.
References
FMC BioProducts RESOLUTIONS, 8(1): 6 - 7, 1992.
Atkins, T., Am. Biol. Teacher, 53: 490 - 495, 1991.
Litz, J.S., FMC BioProducts RESOLUTIONS, 6(3): 6, 1990.
Sakakibara, R., et al., Anal. Biochem., 162: 150 - 155, 1987.
Morgan, J. H., et al., BioTechniques, 11: 256 - 261, 1991.
Coligan, J.E., et al., Current Protocols in Protein Science, Wiley & Sons, Inc., 2001.
Kim, R., et al., Analytical Biochem., 282(1): 147 - 149, 2000.
Wu, M. and Kusukawa, N., BioTechniques, 24: 676 - 678, 1998.

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