Lonza Rockland, Inc.
www.lonza.com
biotechserv@lonza.com
Tech Service: 800-521-0390
Customer Service: 800-638-8174
Document # 18644-1007-04
Rockland, ME 04841 USA

IsoGel® Agarose
Agarose for Isoelectric Focusing
Introduction®

Reagents Required

IsoGel Agarose for isoelectric focusing (IEF) is highly purified
and specially manufactured to yield unmeasurably low®
electroendosmosis (EEO). An IsoGel Agarose gel is easy to
prepare and produces a gel with high clarity and less restrictive
matrix than polyacrylamide, thereby allowing rapid focusing of
higher molecular weight proteins (≤200 kD). Focused proteinscan be transferred from the porous agarose matrix or detectedby immunological techniques. It is also possible to transfer
proteins from these agarose gels into a second dimension
polyacrylamide or agarose sieving gel.

•
•
•
•
•

®

IsoGel Agarose
Ampholytes
d-sorbitolpI markers
Electrolyte solutions

Electrolytes
When selecting anolytes and catholytes for any particular pH
gradient, it is important to closely bracket the ends of the pH
range of the ampholytes. Avoid creating pH discontinuities
between the ends of the ampholyte ranges and the bracketing
electrolytes.

®

The porous nature of IsoGel Agarose means you can readily
fix, stain and destain your gel in a fraction of the time needed®
for polyacrylamide IEF. IsoGel Agarose gels have very low
background stain and proteins are easily visualized byconventional protein stains.
®

IsoGel Agarose IEF gels are sufficiently rigid to allow castingof vertical tube gels in addition to vertically molded andhorizontally open cast thin gels. Gels cast on GelBond®
Support Film remain adhered to the film from the time ofcasting through isofocusing, staining and destaining. Agarose®
gels cast on GelBond Film dry down quickly and easily totransparent, permanent records suitable for scanning or
mounting in a laboratory notebook. A modified Silver Stain
procedure has been developed for use with agarose gels cast®
on GelBond Film.

AnolytePhosphoric acidSulfuric acidAcetic acidL-glutamic acid
Indole acetic acidL-tyrosine

Concentration
1M
0.2 M
0.5 M
40 mM
3 mM
4 mM

pH (25ºC)*
1.0
1.6
2.6
3.2
3.8
4.5

Catholyte
Threonine
GlycineConcentration
50 mM
50 mM

pH (25ºC)*
5.8
6.15

Hepes
0.4 M
7.3
L-Histidine (free-base)**
40 mM
7.35
Bicine0.1 M
8.0
Sodium hydroxide
1M
13.0*pH is dependent on temperature. 25ºC pH values areprovided for selection of electrolytes. Underrunning conditions the pH will be slightly higher.
**Do not substitute Histidine HCL for free-base.

A technique for direct tissue isoelectric focusing (DTIF) of®
soluble proteins has been developed for IsoGel Agarose
whereby tissue samples can be applied directly to the gel
without the need for extraction.
®

IsoGel Agarose IEF gels are compatible with immunofixation,
crossed immunoelectrofocusing and preparative IEF
techniques.

IsoGel® Plate Preparation
Apparatus Required
•
•
•
•
•
•
•
•
•
•

®

The following example details the preparation of a 1% IsoGel
Agarose gel, 0.8 mm thick, cast on a 110 mm x 125 mm sheet®
of GelBond Film. The total solution volume to be prepared is®
10 ml. Alternatively, a larger IsoGel Agarose solution may be
prepared (without ampholytes) and stored refrigerated in tightly
sealed aliquots for later remelting, addition of ampholytes, andcasting into single gels.

Isoelectric focusing chamber
Constant power supply
Refrigerated circulator bath
Forced air oven
Sample applicator mask
Blotting paper
Electrode wicks®
GelBond Film, 110 mm x 125 mm
1-cc tuberculin syringe
20-cc syringe with a large gauge needle14.

Gel Casting Assembly
Assemble the materials required for gel casting
•
•
•
•

5.

Two thick glass plates (size 110 mm x 125 mm)®
One sheet of GelBond Film, 110 mm x 125 mm
A plastic 0.8 mm thick U-frame spacer or 3 single spacers,
0.8 mm thickSix stationery binder clamps

For either dissolution method we recommend taring the
beaker and solution before boiling. After dissolution, add
sufficient warm distilled water to obtain initial weight and mix.
Cool the solution to approximately 60ºC.

Casting Cassette Assembly
1.
2.

3.

4.

5.

Remove the container from oven and GENTLY swirl toresuspend settled powder and gel pieces. Reheat on
HIGH (100%) power setting for 1-2 min., or until thesolution comes to a boil.
Remove from microwave and GENTLY swirl to thoroughly
mix the agarose solution.

1.

Spread a few drops of distilled water or 0.1% nonionicdetergent on one glass plate.
®
Lay a sheet of GelBond Film, cut slightly smaller than theglass plate, on to the plate with the hydrophilic side up(water droplets spread on the hydrophilic side but bead up
on the hydrophobic side).
Cover the GelBond® Film with a sheet of blotting paper or®
the interleaving paper supplied with the GelBond Filmand firmly roll with a rubber roller or wipe with tissues tosqueeze out any air bubbles and excess fluid from behind®
the GelBond Film. Carefully wipe off any excess liquid atthe edges.
®
Place the U-frame spacer on top of the GelBond Film, or®
place two spacers on the GelBond Film along either side
and one across the bottom edge. Place the second glass
plate over the spacer(s). Clamp the assembly with thestationery clamps using 2 clamps per side and bottom.
Warm the cassette in a 55ºC forced hot air oven for 15
minutes.
®
NOTE: GelBond Film may warp if the cassette is heated
too long or above 75ºC.

2.

3.
4.
5.

6.
7.
8.
9.

Preparation of the Gel Solution

Add 0.63 ml of ampholytes 40% solution with a 1.0 cc
tuberculin syringe to the cooled solution.
Stir the solution well to mix. Maintain the
IsoGel®/sorbitol/ampholyte solution at 60ºC-65ºC until thetime of casting the agarose gel. Correct for evaporation
by adding warm distilled water immediately before
transferring the solution to the casting assembly.
Flush the 20-cc syringe with boiling water to thoroughly
heat it.
Expel all water from the barrel and needle.
Immediately fill the syringe with the warmed agarose
solution, and inject it in a steady stream into the warmedcassette. Try to avoid introducing air bubbles into thecassette.
Fill the cassette to the top with agarose solution.
Seal the top of the cassette with parafilm or tape toprevent evaporation.
Allow the casting assembly to cool at room temperature.
Refrigerate it a 4ºC for one hour.
NOTE: Set the refrigerated circulator bath to 10ºC-15ºC.
Do not circulate the coolant to the IEF chamber until
focusing is about to begin. This prevents condensation on
the gel and platen.

In both the following procedures, add room temperature
distilled water and a magnetic stir bar to a beaker or flask thatis 2-4 times the volume of the solution; sprinkle in the agarose
powder while the solution is stirred to prevent formation ofclumps.

Disassembly of the Casting Assemble

Boiling Water Bath or Hot Plate Procedure

3.

1.
2.

3.
4.
5.
6.

1.
2.

Weigh out separately 0.1 g IsoGel® Agarose and1.0 g d-sorbitol.
Add 8 ml distilled water to the flask or beaker and slowly
sprinkle the agarose powder into the distilled water withconstant stirring. Using a spatula, break up and disperse
any agarose clumps and scrape down any powder
adhered to the walls of the flask.
Add the sorbitol to the beaker with constant stirring.
Record tare weight of the beaker and contents.
Cover the beaker with plastic wrap and pierce a small holein it.
Heat while stirring to 90ºC-95ºC and boil for 10 minutes oruntil the agarose is dissolved.

Preparation for Focusing
WARNING: Make certain the power supply is turned
off before proceeding.
Gel Placement
1.
2.
3.

Microwave Procedure
1.
2.
3.

Remove the tape and all clamps.
With the cassette lying flat, insert a flat spatula between
the glass plates and twist gently to break the seal.
Carefully remove the top plate leaving the gel and the®
GelBond Film attached to the back plate.
®
Remove the spacer(s) and lift the GelBond Film from the®
back plate by inserting a flat spatula under the GelBond
Film and gently lift it away from the glass plate.

Follow steps 1 thru 4 above.
Remove the magnetic stir bar. Cover the flask with plastic
wrap and pierce a small hole.
Heat in the microwave oven on MEDIUM (70%) power
setting for 2 minutes.

4.

2

Spread a small volume (0.2 ml-0.3 ml) of distilled water on
the cooling platen of the IEF chamber.
Lower the gel on the wetted area. Avoid trapping air®
under the GelBond Film. Wipe excess fluid from theedges of the film.
Blot the surface of the gel briefly with a sheet of finegrained blotting paper.
If necessary, trim the edges of the gel parallel to thedirection of focusing with a razor blade to ensure that theedges are even and free of cracks or small tears.

5. Place the electrodes on the wicks (not the gel surface),aligning them so that they are parallel to the gel.
6. Set the power supply at 1 W (constant power) and apply
power for 10 minutes.
7. Turn off the power and remove the sample applicatormask. The applicator mask is reusable and should be
rinsed with distilled water.
8 .Gently remove any precipitated sample from the gel surface
with blotting paper.

Electrode Wick Application
1.
2.

3.
4.

5.

Cut two electrode wicks to the exact width of the gel orslightly shorter.
Using forceps to hold one end of an electrode wick,immerse it completely in the appropriate catholyte
solution. Remove excess fluid by placing the wick onblotting paper. This light blotting facilitates the uptake ofany surface moisture which might accumulate near thecathode during prolonged focusing.
Repeat this dipping procedure on a second wick using theappropriate anolyte solution for the anodal wick.
Place the blotted anodal wick on the gel surface at the (+)
electrode contact of the gel. Place the cathodal wick onthe (-) electrode contact of the gel. The wicks must lie
parallel to each other on the ends of the gel, evenly
touching the surface.
Lay a glass plate, slightly larger than the gel, on top of thegel and wicks for approximately one minute. This ensuresuniformity of contact between wicks and gel and serves tosmooth the wick surface in preparation for electrodecontact.

IEF Power Settings and Focusing Time
1.
2.
3.

4.

Set the voltage, current and power according to theappropriate running conditions listed below.
Start circulation of the coolant to the IEF chamber.
The separation progress can be monitored by observing
the visible proteins in the pI markers focus and the rapid
drop in current on the power supply’s milliampere
indicator. Focusing is attained when the visible pl markers
are sharply resolved and the current has stopped
decreasing significantly (less than 1 mA in 10 minutes).
Once focusing is complete, turn off the power, discard thewicks and place the gel in fixative.

Sample Application
1.

Place the sample applicator mask across the gel at least
1 cm from either wick (e.g. 3 cm from cathode).
2. Before applying the sample care must be taken that salts
are removed from the sample by dialysis against water,
0.05 M ammonium bicarbonate or 1% glycine.
3. Pipet the sample solutions into the slots (2μl-5 μl .
maximum; 2-10 mg/ml concentration). In direct tissue
isofocusing tissue samples may be placed directly onto theapplicator slots. pI markers should be run on each gel toprovide stainable markers on known isoelectric points andto indicate progress during the run.
4. Be sure that the electrodes and the electrical contacts areclean and that there are no breaks in the wire or ribbon.

Running Conditions
PH
Range

Voltage(limiting)

2.5-4.5

250 V

3.5-9.5
3.5-9.5

500 V

3.59.54-6.5
5-8

Current

AnolyteCatholyte

MAX

Power
(upperlimiting)
10 W

1 M H3PO4

0.05 M Threonine

Focusing
Time(Minutes)
90

1000 V
1500 V

MAX
MAX
MAX

10 W
25 W
25 W

0.5 M HOAc
0.5 M HOAc
0.5 M HOAc

1 M naOH
1 M NaOH
1 M NaOH

90
40
30

500 V

MAX

10 W

0.1 M Bicine

90

500 V

MAX

10 W

2% solution of
pH 2.5-4.5
Ampholyte
2% solution of
pH 2.5-4.5
Ampholyte

0.1 M NaOH or
0.1 M LHistidine(free-base)90*1 M H3PO4 can be replaced by 0.5 M acetic acid3

Fixing & Staining Solutions
Fixative solution

Stop Bath1%
Acetic acid180 mlMethanol
30 g
Trichloroacetic acid18 g
Sulfosalicylic acid
Fill to 500 ml with distilled water*available from Sigma Chemical Co.

Fixing, Staining and Destaining
Coomassie® Stain or Crowle’s Double Stain

180 mlMethanol
30.0 gTrichloroacetic acid18.0 gSulfosalicylic acid
Fill to 500 ml with distilled water

The following protocol applies to either stain
1. Place the gel in fixative for 10 minutes.
2. Grasp an edge of the gel film support with forceps to lift
the gel from the fixative.
3. Rinse the gel surface with distilled water and drain excess
solution.
4. Place the gel on a paper towel, gel side up.
®
5. Place a single sheet of Whatman 3MM Blotting Paper,wetted with distilled water, on the gel surface.
6. Overlay the blotting paper with six layers of absorbent
paper toweling.
7. Place a glass plate on top of the paper toweling andweight it down with a 1 kg-2 kg weight for 10 minutes.
8. Remove the weight, the glass plate, and the paper
toweling.
9. Rewet the blotting paper thoroughly with distilled waterand gently lift it off the gel surface.
10. Wash the gel in distilled water for 15 minutes to remove
residual fixative and ampholytes.
11. Dry the gel completely in a forced hot air oven (50ºC55ºC). Drying usually takes less than ½ hour.
®
12. Stain with Coomassie or Crowle’s Double Stain for 15-30
minutes.
13. Float the gel face down into the stain so that precipitatedstain will not settle on the gel surface.
14. Remove the gel, rinse it with distilled water.
15. Place the gel in destain for 3 minutes.
16. Rinse again briefly in distilled water. (This applies for
either stain used).
®
17. Clamp the GelBond Film, gel side out, onto a glass plate
to prevent curling during drying.
18. Dry the gel either in a forced hot air oven (50ºC-55ºC) for
approximately 15 minutes or dry at room temperature
overnight.
NOTE: Gels will crack if overdried. The gel can be read,stored or otherwise used without further treatment.

Pretreatment solution 1

Silver Stain

2% glutaraldehyde

A modified silver stain procedure has been developed for use®
with agarose gels cast on GelBond Film. After electrophoresis
the gels are fixed, press blotted and completely dried down
before staining. Perform all fixing and staining steps in acidcleaned (50% HNO3) glassware. All washes are done withconstant agitation in a volume of at least 250 ml (gel®
volume:reagent volume = 1:22). Coomassie Brilliant Blue
stained gels may be silver stained after drying. In this caseproceed to the glutaraldehyde pretreatment step (below).

Either Coomassie® Blue Stain or Crowle’s Stain can be used to®
stain IEF gels. Coomassie Stain is used when increased
sensitivity is desired, and Crowle’s Double Stain produces gelswith clear background and sharp resolution.

Coomassie® Stain®
1.0g
Coomassie Brilliant Blue R-250
250 mlEthanol
90 ml
Glacial acetic acid
Fill to 1 liter with distilled water

Coomassie® Destaining Solution
250 mlEthanol
90 ml
Glacial acetic acid
Fill to 1 liter with distilled water

Crowle’s Double Stain
2.5 g
Crocein Scarlet (C.I. 26905)®
150.0 mg
Coomassie Brilliant Blue R-250
50.0 ml
Glacial acetic acid30.0 gTrichloroacetic acid
Fill to 1 liter with distilled water

Crowle’s Destaining Solution
3 ml
Glacial acetic acid
Fill to 1 liter with distilled water

Silver Stain
A modified Silver Stain procedure has been developed for use®
with agarose gels cast on GelBond Film.

Fixative solution

Pretreatment solution 2
0.01% DTT (dithiothreitol)

Silver Stain Solution A
Sodium carbonate, anhydrous
50.0 g in 1 L distilled water(Stable for 2-3 weeks at room temp.)

Silver Stain Solution B

1.
2.
3.

Combine the following solutions into 1 liter of distilled water,while mixing rapidly, in the order given:
2.0 g ammonium nitrate, 2.0 g silver nitrate, 10.0 g dodecatungstosilicic acid* and 6.7 ml 37% formaldehyde.
(Stable 1 week at room temp. Stored in the dark).

4.
5.

4

Fix the gel for 10 minutes.
Place the gel on a paper towel, gel side up.
Place a single sheet of Whatman® 3MM Blotting Paper,wetted with distilled water, on the gel surface.
Overlay the blotting paper with six layers of absorbent
paper toweling and weight it down with a 1 kg-2 kg weightfor 30 minutes.
Remove the weight, the glass plate, and the paper
toweling.

6.

Rewet the blotting paper thoroughly with distilled waterand gently lift it off the gel surface.
7. Wash the gel in distilled water for 15 minutes to remove
residual fixative and ampholytes.
®
8. Clamp the GelBond Film, gel side out, onto a glass plate
to prevent curling during drying.
9. Dry the gel in a forced hot air oven (50ºC-55ºC) for
approximately 15 minutes or until dry.
NOTE: Gels will crack if overdried.
10. Soak the dried gel in 2% glutaraldehyde for 10 minutes,
followed by a 10 minute wash in distilled water using mild
agitation.
11. Soak the gel for 10 minutes in 0.01% DTT(dithiothreitol),followed by a 10 minute wash in distilled water using mild
agitation.
12. Pour an equal volume of Silver Stain Solution B into
vigorously stirring Silver Stain Solution A (75 ml B and
75 ml A for each gel to be stained).
13. Transfer the solution to an acid-cleaned glass dishcontaining one gel.
14. Stain the gel 10 minutes with gentle agitation. (There will
be some background).
15. Transfer the gel to a stop bath containing 1% acetic acidand gently agitate for 5 minutes.
16. Rinse the gel in distilled water, wipe any silver deposits
from the back of the gel and allow to air dry.

Transfer of proteins from the IEF gel to nitrocellulose:
1. Prepare a solution of Tris-saline buffer, pH 7.5:
50.0 g Tris-HCLl0.94 g Tris-Base58.48 g NaCl
Fill to 2 liters with distilled water.
2. Cut one piece of nitrocellulose membrane and thick filter
paper to the same dimension as the IEF gel. (Weargloves to prevent contamination by extraneous proteins).
3. Evenly wet the nitrocellulose membrane in the Tris-saline
buffer by holding one end of the membrane with smoothtipped forceps and lowering the other end into the buffer
container, dropping the membrane flat on the buffer
surface. The membrane must be completely saturated
with buffer.
®
4. After isoelectric focusing is complete, remove the IsoGel
Agarose IEF gel from the chamber and place it on a flat
surface, gel side up.
5. Place the buffer-soaked nitrocellulose membrane onto thegel surface. Avoid trapping air bubbles between the gel
and the membrane.
6. Place one piece of buffer soaked filter pad on top of thenitrocellulose membrane followed by three layers of dry
paper toweling.
7. Cover with a glass plate slightly larger than the gel
surface. No other weight is used.
8. Press blot for 1 ½ minutes.
9. Remove glass plate and discard the paper toweling andfilter paper.
10. Transferred proteins can be detected on the membraneand on the gel by standard methods. Differential detection
may be done.

Sample Pretreatment
Successful isoelectricfocusing in part depends upon thecondition of the sample. Situations such as high salt content,
particularly in cases of high sample loading, or insolubility
should be addressed before the sample is loaded onto the gel.
•
Dialyze the sample against distilled water, 1% glycine or
0.05 M-0.1 M ammonium bicarbonate solution for samples
with high salt content.
•
To dissociate protein aggregates, or subunit assemblies
and to unfold peptide chains: Add urea to a final
concentration of 4 M to 9 M to both the sample and thegel.
•Samples that are hydrophobic or poorly soluble at or near
their pI point.
•
Add either nonionic or zwitterionic detergents to thesample and the gel at a final concentration of 0.05-1.0%.
Nonionic detergents®
Triton X-100
Nonidet® (NP-40)®
Tween 80
Zwitterionic detergents®
CHAPS(available from Sigma Chemical Co., St. Louis, MO)®
Zwittergent 3-14
(available from Calbiochem/Behring, LaJolla, CA)

Preparative Isoelectric Focusing
Separation of relatively large amounts of biologically active
macromolecules is possible by isoelectric focusing in agarose.
Typical high yield recoveries of applied proteins are obtainablewith retention of biological activity. This preparative isoelectric
focusing procedure is based on the work of Cantarow, et al.
As much as 120.0 mg of protein can be focused in 9.5 ml (0.75®
cm x 10 cm x 11.5 cm) of 1% IsoGel Agarose containing 2.5%®
ampholytes. Follow the procedure described under IsoGel
Plate Preparation for gel casting adding the sample to the
IsoGel® Solution at 60ºC.
After gelation, allow the gel to equilibrate before beginningisofocusing by placing it on the 10ºC cooling platen for 10
minutes.
Run the isoelectric focusing under standard conditions. To
recover proteins from the gel use a spatula to cut 5 mm wideslices from the gel, stripping the agarose from the GelBond®
Film. Place the agarose strip in a 5-cc plastic syringe fittedwith an 18-gauge needle. Macerate the gel slice by expelling it
into a clean tube. Add 4.0 ml of phosphate-buffered saline(PBS) to the macerated gel. Cover the tubes and place themon a test tube rocker for 16 hours at 4ºC. Centrifuge themixture for one minute at 100 x g and separate the supernatantfrom the gel using a serum separator.

Press Blot Transfer of Proteins from IsoGel®
AgarosePress blot transfer is a quick method of removing proteins from
agarose gels. The procedure involves overlaying the gel with abuffer-soaked nitrocellulose membrane covered by a thick filter
pad and several layers of dry paper toweling. The assembly isthen covered by a glass plate. After just 1 ½ minutes of press
blot approximately 20% of the proteins are transferred from thegel to the membrane. Up to 85% transfer can be achieved witha 35-40 minute blotting time.

Immunofixation/Immunoperoxidase orAutoradiography
Detection of separated species can be accomplished byprotein stains or by overlaying gels with specific antibody
solutions coupled to enzymes that will eventually produce avisible end product. The antibody-peroxidase conjugate

5system or autoradiography with 125I labeled antibody are
frequently employed for this purpose.

agarose gelation and the gels that are formed are much
weaker and more fragile than standard IEF gels. Therefore, it
is best not to blot an agarose-urea gel. After gelation, gels
containing urea should be refrigerated overnight before use.

Immunoperoxidase labeling of focused proteins is done
according to Saravis, et al. After focusing, fixing and drying the
IEF gel, treat the gel as outlined below:

Gel Preparation
Agarose-urea gels should be prepared by first dissolving the
agarose in a minimum volume of water or buffer, then coolingto slightly below 50ºC before stirring in solid urea or a highconcentration solution of urea. Once the urea has dissolved,
adjust to the desired total volume with water or buffer. Casthigh urea concentration gels (7 M-9 M) and refrigerate
overnight. If gels are quick-chilled by placing them in a freezer
for 15-20 minutes, urea crystals may be removed by allowing
the gel to sit at room temperature for a few minutes.

Immunoperoxidase Staining/Avidin-Biotin Modification:
1.
2.
3.
4.
5.
6.
7.
8.

Soak the gel in 3% hydrogen peroxide for 10 minutes.
Rinse with distilled water.
Soak the gel in 2.28% periodic acid for 5 minutes.
Rinse with distilled water.
Soak the gel in 0.02% sodium borohydride for 2 minutes.
Rinse with distilled water.
Place the gel in Tris-saline 0.05 M, ph 7.6 for 10 minutes.
Incubate the gel with 1:5 normal serum for 10 minutes(same species as secondary antibody).
9. Incubate the gel for 2-4 hours at room temperature with
anti-(primary antibody).
10. Rinse the gel with 0.15 M phosphate-buffered saline(PBS), pH 7.4, for 1 hour at room temperature.
11. Incubate the gel with secondary* antibody (e.g. Goat antimouse IgG). If the avidin/biotin modification is used,
proceed with steps 12 and 13 using biotinylated reagents(marked with * in steps 11 and 15).
12. Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.
13. Incubate the gel with avidin solution (40 mg/gel) in PBS for
1 hour at room temperature (stock solution of avidin is
usually 20 mg/ml).
14. Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.
15. Incubate the gel with horseradish-peroxidase* (33 mg/gel)for 2 hours at room temperature.
16. Rinse the gel with 0.15 M PBS for 1 hour at room
temperature.
17. Incubate the gel with diaminobenzidine (0.15 mg/ml) andhydrogen peroxide (0.03%) in
0.01 M Tris-0.15 M NaCl for 1-17 hours at room
temperature.
18. Rinse the gel in PBS and dry.

®

Gel strength may be enhanced by increasing the IsoGel
Agarose concentration of the gel. If a satisfactory gel cannot
be obtained by the above procedure, cast an aqueous IsoGel
Agarose gel, and then allow it to equilibrate with a urea/
ampholyte solution.
CAUTION: Avoid exposing urea to high temperatures. Urea
solutions decompose to ammonia and carbon dioxide withexcessive heat. Cyanate formation and carbamylation of othergel components, such as ampholytes, may also occur.

Bibliography
General1.
Harper, D.L. “Isoelectric focusing in thin-layer agarosegels”. Electrophoresis ’81, 205-212. Edited by Allen,
R.A. and Arnaud, P., Berlin: de Greater and Co., 1981.
2.
Coulson, SE “Enhancement of complex patterns inisoelectric focusing with improved IsoGel performance”.
Electrophoresis ’81, 229-236. Edited by Allen, R.A. andArnaud, P., Berlin: de Gruyter and Co., 1981.
3.
Saravis, C.A., and Zamcheck, N. “Isoelectric focusing inagarose”. J.Immunol.Methods 1979, 29: 91-96
4.
Hjelmeland, L.M., and Chrambach, A. “Electrophoresisand electrofocusing in detergent containing media: A
discussion of the basic concepts”. Electrophoresis
1981, 2: 1-11.
5.
Rilbe, H., “Historical and theoretical aspects of isoelectric
focusing”. Ann. N.Y. Acad. Sci. 1973, 209: 11-22.
6.
Radola, B.J., and Graesslin, D., (eds.), “Electrofocusing
and isotachophoresis”. Berlin: De Gruyter and Co., 1977.
7.
Catsimpoolas, N., and Drysdale J., (eds.), “Biological andbiomedical applications of isoelectric focusing”. Plenum,NY, 1977.
8.
Vesterberg, O., “Isoelectric focusing: A review ofanalytical techniques and applications”. Int. Lab. 1978,
May/June.
9.
Malamud, D., and Drysdale, J.W., “Isoelectric points ofproteins: A table”. Biochem. 1978, 86: 620-647.
10. Righetti, P.G., and Caravaggio, T., “Isoelectric points andmolecular weights of proteins. A table”. J.Chromatog.
1976, 127: 1-28.
11. Righetti, P.G., Tudor, G., and Ek,K. “Isoelectric points
and molecular weights of proteins. A new table”. J.
Chromatog. 1981, 220: 115-194.

Direct Tissue Isoelectric Focusing (DTIF)
This method employs the application of tissue or cell pellets
directly onto the surface of the IEF gel without concentration ofsamples, dialysis to remove salts, or the salt extraction ofsoluble proteins from tissue. Briefly, a tissue slice is draped
into the open slot of an application mask on the gel surface,refocused for 10-15 minutes at low voltage to allow sample
uptake, and then completely removed. Focusing is continued
using standard methods. After focusing is complete, the gel isfixed, stained and dried using standard conditions.
DTIF allows more soluble protein per milligram of tissue toenter the gel than is recoverable by extraction procedures andminimizes denaturation of biologically active proteins that canbe damaged by extraction.

Urea in IsoGel® IEF Gels
Urea is a common additive in the focusing of membrane andaggregate proteins. High concentrations of urea inhibit

6Special Techniques
12. Cantarow, W.D. Saravis, C.A., and Ives, D.V., et al.
“Preparative electrofocusing in agarose slab gels”.
Electrophoresis 1982, 3: 85-89.
13. Saravis, C.A., Cantarow, W., and Marasco, P.V., et al.
“Amplified immunoperoxidase staining of isoelectricallyfocused human tumor markers”. Electrophoresis 1980,
1: 191-193.
14. Saravis, C.A., O’Brien, M., and Zamcheck, N. “Direct
tissue isoelectric focusing in agarose”. J.Immunol.
Methods 1979, 29: 97-100.
15. Ebers, G.C., Rice, G.P., and Armstrong, H. “Preparative
isoelectric focusing in agarose”. J Immunol. Methods
1980, 37: 315-323.
16. Emerson, D.L., Chapins-Cellier, C., and Arnaud, P.
“Horizontal two-dimensional(ISO-DALT). Electrophoresis:
Use of agarose isoelectric focusing and SDS gelelectrophoresis in an exponential gradient forcharacterization of plasma proteins”. Electrophoresis
1980, 1: 159-163.
17. Coulson, S.E., and Cook, R.B. “Two-dimensional
electrophoresis in agarose gels”. Electrophoresis ’82.
Edited by Stathakos, D., Berlin: de Gruyter and Co.,1982.
18. Geoghegan, W.D., Williams, D.L., and Jordon, R.E. “Theapplication of glucose oxidase-labeled antibodies for thedetection of proteins on nitrocellulose”. J.Immunol.
Methods 1986, 93: 231-236.
19. Gunzer, G. “Titration curves in ultrathin polyacrylamide
and agarose gels”. Electrophoresis 1984, 5: 370-372.
20. Van Triet, A.J. “Ultrathin-layer isoelectric focusing inagarose gels and visualization of urinary alanine
aminopeptidase with a modified agar overlay technique:.
Electrophoresis 1984, 5: 374-376.
Applications
21. Klose, J. “Isoelectric focusing and electrophoresis
combined as a method for defining new point mutations in
the mouse”. Genetics 1979, 1: 13-24.
22. Rosen, A., Ek,K., and Amen, P. “Agarose isoelectric
focusing of native human immunoglobulin M. and alpha2-macroglobulin”. J.Immunol. Methods, 1979 28: 1-11.
23. Vesterberg, O., and Holmberg, K. “Characterization ofallergen extracts by two-dimensional electrophoretic
techniques: Micropolyspora faeni antigen”. Clin. Chem.
1982, 28: 993-997.
24. Lundstrom, R.C. “Rapid fish species identification byagarose gel isoelectric focusing of sarcoplasmic
proteins.” J. Assoc. Off. Anal. Chem. 1981, 64: 38-43.
25. Richetti, P.G., and Bosisio, A.B. “Application of isoelectric
focusing to the analysis of plant and food proteins”.
Electrophoresis 1981, 65-75.
26. Binion, S., and Rodkey, L.S. “Simplified method forsynthesizing ampholytes suitable for use in isoelectric
focusing of immunoglobulins in agarose gels.”
Anal.Biochem. 1981, 112: 362-366.
27. Steinitz, M., Izak, G., and Cohen, S., et al. “Continuous
production of monoclonal rheumatoid factor by EBVtransferred lymphocytes”. Nature 1980, 287: 443-445.
28. Dykes, D.D., DeFurio, C.M., and Polesky, H.F.
“Transferrin (Tf) Subtypes in US Amerindians, Whites andBlacks using thin-layer agarose gels: Report on a new
c8variant Tf ”. Electrophoresis 1982, 3: 162-164
29. Dykes, D.D., Copouls, B.A., and Polesky, H.F. “Routine
phenotyping of phosphoglucomutase (PGM¹) by thin-layer

30.

31.

32.
33.

34.

35.
36.
37.

38.
39.

7

focusing: Isoelectric points of 14 different varients”.
Electrophoresis 1982, 3: 165-168.
Kostulas, V.K., and Link, H. “Agarose isoelectric focusingof unconcentrated CSF and radio-immunofixation for
detection of oligoclonal bands in patients with multiple
sclerosis and other neurological diseases”. J.Neurol.
Sciences 1982, 54: 117-127.
Kalsheker, N.A., Warner, J.T., and Watkins, G.L.
“Phenotyping alpha-1-antitrypsin variants by isoelectric
focusing in agarose and immunoblotting”. Clin. Chim
Acta 1985, 148: 157-160.
Hoffman, W.L., Douglass, R.R., and Smiley, J.D.
“Synthesis of electrophoretically restricted IgG by cultured
rheumatiod synovium”.Arthritis Rheum 1984,27:976-984
Lane, J.R., Bowels, K.J., and Normansell, D.E. “Detectionof oligoclonal lgG bands in cerebrospinal fluid by
isoelectric focusing on thin layer agarose gels”.
Ann.Clin.Lab.Sci. 1984, 14: 146-150.
Qureshi, A.R., and Punnett, H.H. “An improved method
for phenotyping of alpha 1-antitrypsin variants using
separator isoelectric focusing on thin-layer agarose gel”.
Anal.Biochem. 1982, 125: 335-338.
Wiggin, K., Krzynowek, J. “Identification of frozen cookedshellfish species by agarose isoelectric focusing.”
J.Assoc. Off.Anal.Chem. 1983, 66: 118-122.
Wilson, C.M. “Isoelectric Focusing of zein in agarose”.
Cereal Chem. 1984, 61: 198-200.
Rostrom, B., Boe, J., and Lilja, I. “Demonstration of
intrapleural immunoglobulin synthesis by agarose gel
isoelectric focusing”. Am. Rev. Respir. Dis. 1986, 134:
1036-1039.
Whitmore, D.H. “Parvalbumins for calibrating acidic
isoelectric focusing gels”. Anal. Biochem. 1989, 178:
121-124.
Chiodi, F., Siden, A., and Osby, E. “Isoelectric focusing of
monoclonal immunoglobulin G,A and M followed bydetection with the avidin-biotin system”. Electrophoresis
1985, 6: 124-128.

For Laboratory Use.
Derived from Agar.

Ordering Information:
Catalog No.
Description
50202
IsoGel® Agarose

Size
25 g

Related Products:
The IsoGel® Accessory Kit
The IsoGel® Agarose IEF Plate Accessory Bulk Pack
GelBond® Film
For more information contact Technical Service at(800) 521-0390 or visit our website atwww.Lonza.com.

.

GelBond is a trademark of FMC Corporation. Coomassie is atrademark of ICI,Lourdes. Triton is a trademark of Rohm & Haas Co.
Whatman is a trademark of Whatman, Inc., Tween is a trademark of
ICI Americas Inc., Zwittergent is a trademark of Hoechst CelaneseCorp and Nonidet is a trademark of Shell. All other trademarks herein
are marks of the Lonza Group or its affiliates.
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