Lonza Rockland, Inc.
www.lonza.com
scientific.support@lonza.com
Scientific Support: 800-521-0390
Customer Service: 800-638-8174
Document # 18657-0111-06
Rockland, ME 04841 USA

GelBond® PAG Film

Product Stability
GelBond® PAG Film is sensitive to heat and light. It ispackaged in a light-barrier wrapper, and if stored in adrawer at room temperature (<26οC), will remain stable
for at least two years from date of manufacture.
Although packages are marked “light sensitive”,
exposure to room lighting for a few hours does notdeactivate the coating. Once a gel is cast on GelBond®
PAG Film, the film is no longer sensitive to light.

Introduction
GelBond® PAG Film is a patented, transparent, flexible
polyester film designed to support polyacrylamide, Long
Ranger™, ProSieve™ 50, or MDE™ Gels1. The
acrylamide monomers covalently attach to the coatingon the film during the polymerization process so thatslab gels of commonly used acrylamide concentrations
can be easily handled without tearing. Gels remain
permanently attached to the film through electrophoresis
and all subsequent fixing, staining, destaining and drying
procedures. Gels retain their original dimensions(except thickness) after drying. Dried gels are suitablefor densitometric scanning, autoradiography, projection
or permanent mounting in laboratory notebooks.

Mounting GelBond® PAG Film in Casting Apparatus
Gels must be cast on the treated, hydrophilic side.
GelBond® PAG Film is packed with the treated side up
with paper interleaving to protect the treated surface. If
uncertain, the hydrophilic side can be confirmed byapplying a drop of water and noting that it spreads on
the hydrophilic side, but beads up on the hydrophobicside. Always handle GelBond® PAG Film by the edges
or with gloves to avoid getting fingerprints on the treatedsurface.

GelBond® PAG Film can be used in denaturing andnondenaturing electrophoresis and isoelectric focusing
techniques. However, the 0.2 mm thick polyester film isneither electrically conductive nor porous, so it cannot be
used in electrophoretic destainers or in electrophoretic or
capillary blotting techniques. Gels can be stripped off
GelBond® PAG Film by using a thin wire or cheese
cutter2.

Studier-Design Chambers
Studier-design3 electrophoresis chambers use a notchedglass plate to connect the upper buffer reservoir with thegel. In these chambers, GelBond® PAG Film should be
mounted on the unnotched glass plate. Substitute forchambers a shorter plate for a notched plate. Mount
GelBond® PAG Film onto the larger-outer plate. The film
should be the same size as the unnotched glass plate orjust slightly smaller.

GelBond® PAG Film will fluoresce when exposed to UV
light. This background fluorescence may interfere withvisual detection of fluorescent proteins or nucleic acids
stained with ethidium bromide. If photography is used,background fluorescence can be screened out by using
red, orange (e.g. Wratten® Gelatin Filter), and UV
camera filters. GelBond® PAG Film will not transmit lightof less than 310 nm, so nucleic acid gels stained withethidium bromide should be inverted (gel side down)
onto the light box in order to expose the separated
sample bands to full light intensity. The fluorescing
nucleic acid bands can be photographed through
GelBond® PAG Film using this method.

1. Place a few drops of distilled water, 0.1% Triton® X100 nonionic detergent, or 5% glycerol solution on
the unnotched glass plate.
2. Without getting fingerprints on the GelBond® PAG
Film, lay a sheet squarely on the glass plate, withthe hydrophilic side facing up.

GelBond® PAG Film can be used with bisacrylamide(N, N‫ ׳‬-methylene-bis-acrylamide). With bisacrylamide,
the use of a vacuum slab dryer is necessary in order todry gels above 7%-8% T without cracking.

3. With the paper interleaving over the GelBond® PAG
Film, wipe or roll (with a rubber roller) firmly fromthe center to the edges to squeeze out excess fluid
and air bubbles. Wipe up any fluid squeezed out at

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f. Lay the spacers in place with the greased side on
the GelBond® PAG Film, pressing down at the top tosqueeze the dabs of grease from step e. Wipe off
excess grease at the top. Apply a thin line of grease
down the top side of each spacer.

the edges. Remove the paper interleaving andclean off any fluid from the GelBond® PAG Filmsurface. The film must be tightly adhered to theglass plate.
4. Lay spacers on the GelBond® PAG Film andassemble the casting cassette as usual. From thispoint on, gel casting is the same as without
GelBond® PAG Film.

g. Trim off the excess tape, lay the second glass
plate down properly, and assemble the moldaccording to the chamber’s instructions. Tighten all
clamp screws only tight enough to get a good seal
with the greased spacers. Over tightening cancause plates to crack.

Hoefer-Design Chambers
Owners of Hoefer SE design4 series verticalelectrophoresis chambers with cam-gasket seals, suchas those sold by Hoefer, should use the following
procedure to assure tight adhesion of GelBond® PAG
Film in the cassette. This Scotch® Tape procedure,developed by Rosenblum5, has allowed his lab to run
hundreds of gels without leakage.

h. Before camming the mold into the casting stand(or upper buffer tank, later), place dabs of grease on
the corners of the glass plate and spacer ends toprevent leakage. (A light coating of grease on therubber gasket is also helpful.)
3. Insert the comb at a slight angle away from the
GelBond® PAG Film surface. This will align the teeth
and prevent catching them on the edge of the film.
Insert the comb prior to filling the casting assembly
to prevent solution from leaking behind the GelBond®
PAG Film.

1. GelBond® PAG Film should be cut slightly smaller
than the dimensions listed for the glass plates. Thisis important to prevent the film from overlapping theglass plates, which can lead to buckling by the sideclamps or the rubber gaskets at the top and bottomof the apparatus.

Processing Bisacrylamide Gels
Vacuum Drying of High-Concentration
Bisacrylamide Cross-Linked Gels.
To dry bisacrylamide cross-linked gels attached to
GelBond® PAG Film on a vacuum slab dryer, use thefollowing procedure.

2. Use the following sequence to place GelBond® PAG
Film on the glass plate.
a. Cut a strip of double-sided Scotch® Tapeapproximately two inches longer than the width of
the glass plate.

a. Cover the glycerol-soaked gel with a prewet sheet of
porous cellophane (available from vacuum slab dryer
dealers), dialysis membrane, or chromatography paper.
Plastic food wrap will not work.

b. Holding the tape taut, align it with the bottomedge of the glass and lay it in place. This tape will
attach the GelBond® PAG Film to the glass andprevent acrylamide solution from channeling behindthe film during casting.

b. Place several sheets of chromatography paper on thegel dryer and position the gel over these so that the
GelBond® PAG Film is away from the vacuum source.

c. Put a few drops of water in the center of the glass
plate.

c. Dry the gel under vacuum according to the
manufacturer’s instructions. GelBond® PAG Film canwithstand dryer temperatures of 80oC. Drying times
generally range from about one hour for a 0.75 mm thickgel to about five hours for a 3.0 mm thick gel. Whereas
porous cellophane will adhere to the dried gel
permanently, the chromatography paper can beremoved by soaking the dried gel briefly in distilled waterand gently peeling the paper off.

®

d. Position the GelBond PAG Film (hydrophilic side
facing up) so that the bottom edge is aligned withthe bottom edge of the glass. Using a small roll ofcotton gauze or soft paper tissues, press down firmly
to seal the film to the tape. Press out the excess
fluid so that a very tight seal is formed between theglass and the film. Make sure that no air bubbles orpockets of water remain except a small line of air
along the edge of the Scotch tape. Wipe any water
off the GelBond® PAG Film.

Staining and Destaining Procedures
Coomassie® Brilliant Blue Stain
Destain solution is changed 3-4 times to expedite theprocess. Gels can be left in destain overnight withoutcausing the gel to disadhere from the GelBond® PAG
Film. All gels must be soaked in glycerol-preserving
solution for ≥1.5 hours before drying.

e. Grease the side spacers as follows: Dip the top
ends of each spacer into Cello-Seal™ Grease (orsimilar product) to provide a small dab of grease on
the back side. Apply a thin line of grease down theback side of the spacer (conveniently applied bysyringe or by streaking it on with the rounded end of
a test tube).
2Another approach is to cast the gel without detergent,
then equilibrate it in a bath of detergent, urea andalkaline ampholytes for a few hours.

Silver Stain (Adapted from J.H. Morrissey.6
Washing and staining times need to be adjusted for
differing gel thickness or concentrations, since diffusion
can only occur from the exposed gel surface. In general,
it is best to cast a thin gel (≤1 mm) so that developer andstop solutions can readily diffuse into the gel. This
results in better sensitivity and lower backgroundstaining.

Alkaline Ampholytes
Alkaline ampholytes interact with polymerizing
acrylamide solutions to inhibit gel adhesion to GelBond®
PAG Film. This is believed to be due to amino groups in
the ampholytes competing with acrylamide to bind to thereactive GelBond® PAG Film surface. The interference
can occur with ampholytes above pH 8 in narrow or
wide-range pH gradients.

Compatibility
Periodic Acid Silver Stain
GelBond® PAG Film can be used with the Tsai and
Frasch7 periodic acid silver stain procedure forlipopolysaccharide. It is very important to increase
washing times in this application to prevent highbackground stain deposition in the GelBond® PAG Filmsupported gels.8

Good adhesion can be obtained by using no more than
2.5% w/v ampholytes in the gel, while including higher
than normal levels of ammonium persulfate(0.7-1.0 mg/ml of gel-forming solution) and TEMED(1 μl/ml). Since excess salts can disrupt IEF results, thegel can be soaked in a solution of ampholytes or
prefocused for 15 minutes at 500 V to remove salts
before samples are applied.

Kodavue™ Nickle Stain
GelBond® PAG Film is chemically incompatible with the
Kodak® Kodavue Nickle Stain Kit.9

Immobiline® Gels
Immobilized pH gradient isoelectric focusing (IPG-IEF) isa specialized technique for greatly expanding the scaleof narrow-range IEF. The technique employs Immobiline
Ampholytes (Amersham-Pharmacia Biotech) and
GelBond® PAG Film. Immobilized pH gradient gels havebeen frequently reported for nondenaturing IEF.
Modifications of the technique have been reported underconditions that solubilize and denature most proteins.
By reducing TEMED and APS, conditions have been
determined which allow adequate time to pour even very
viscous gel solutions containing 4% acrylamide, 8 M
urea, and 2% NP-40. Under these conditions thegel adheres firmly to the GelBond® PAG Film backing.12

SucroseGradient gels containing 15% sucrose as a weighting
solution of the high-concentration end have beenreported to disadhere from GelBond® PAG Film. The
gels cited contained 5%-20% polyacrylamide, crosslinked with bisacrylamide. For best results when castinggels on GelBond® PAG Film, 15% glycerol should be
used instead of sucrose.
Urea
Urea can be used at high concentrations of 8 M with
GelBond® PAG Film and polyacrylamide gels without
seriously interfering with adhesion. However, urea iscommonly employed in conjunction with otheringredients that do reduce adhesion (i.e. nonionicdetergents and alkaline ampholytes).

Trichloroacetic Acid (TCA)
TCA is frequently used as a fixative after IEF or peptide
electrophoresis. High TCA concentrations can damage
gel adhesion to GelBond® PAG Film. Fixatives with 10%
TCA or less can normally be used without problems if
fixing time is no longer than 30 minutes at room
temperature. IEF gels containing other ingredients thatweaken adhesion (such as nonionic detergents) may bedigested off the support even by 10% TCA.

Nonionic Detergents
Triton® X-100 and Nonidet™ P-40 (NP-40) Detergentsare popular in IEF of sparingly soluble proteins. They
diminish gel adhesion to GelBond® PAG Film, apparently
by blocking the reactive surface. (Gel polymerization
proceeds as usual.) Nonionic detergents with urea aresaid to have a similar effect on silanized polyester andother plastic supports.

For peptides, TCA is typically employed at levels as highas 25% which can cause disadhesion in as little as 30
minutes. Gels left in more dilute fixatives (5%) for
several hours to wash out ampholytes may also
disadhere. Refrigeration of the fixative bath andminimizing the TCA concentration and exposure
time can prevent disadhesion.

Poor adhesion at a 2% NP-40 level can be overcome by
reducing the NP-40 level to 0.5% or lower. Reliablebinding of polyacrylamide gels to GelBond® PAG Filmhas been obtained in the presence of 8 M urea with
0.5% NP-40 or the zwitterionic detergent CHAPS,10 byincreasing the catalyst concentration to 2 to 3-fold. BothCHAPS and CHAPSO11 are compatible with GelBond®
PAG Film.

TCA is sometimes included in the solvent for
Coomassie® Brillant Blue G-250 stain. We do notrecommend use of this procedure.

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References
1. Nochumson, S., et al. U.S. Patent 4,415,428,
November 15, 1983, reassigned to CBM Intellectual
Properties, Inc.
2. Curtis, F.P. and Nochumson, S. BioTechniques 8:40-41, 1990.
3. Studier, F.W. J. Mol. Biol. 79: 237-248, 1973.
4. Hoefer, P.S. et al. U.S. Patent 4,290,871, Sept. 22,
1981, assigned to Hoefer Scientific Instruments.
5. Rosenblum, M., M.D. Anderson Hospital, Houston,TX, personal communications, 1984.
6. Morrissey, J.H. Anal. Biochem. 117: 307-310, 1981.
7. Tsai, C.-M, et al. Anal. Biochem. 119: 115-119,
1982.
8. Cotter, P., State Univ. of NY at Buffalo, personalcommunication, 1983.
9. Yudelson, J., Eastman Kodak Company, Rochester,NY, personal communication, 1985.
10. Dunn, M.J., et al. Electrophoresis ‘84, 281-284. V.
Neuhoff (ed.), Verlag Chemie, Deerfield Beach, FL,
1984.
11. Hjelmeland, L.M. Proc. Nat. Acad. Sci. USA. 77:
6368-6370, 1980.
12. Field, D.J., et al. Anal. Biochem. 144: 584-592,
1985.
13. Pino, R.M., et al. Anal. Biochem. 139: 77-81, 1984.
14. Kobayashi, Y., NEN Research Products, Boston,MA, personal communication, 1984.
15. Schrag, J., Univ. of Illinois, Urbana, IL, personalcommunication, 1984.

Agarose-Acrylamide Composite Gels
Neither GelBond® PAG Film nor standard GelBond® Filmis designed for, or reliable with, composite gels. For
composites in which acrylamide is allowed to polymerize
above the gelling temperature of the agarose, GelBond®
PAG Film may adhere the gel better than standard
GelBond® Film.
Conversely, if the agarose is allowed to gel before theacrylamide polymerizes, standard GelBond® Film ispreferable. However, there is less adhesion ofcomposite gels than agarose gels.13
Dehydrating Solutions
Severe shrinkage followed by expansion is often thecause of disadhesion from GelBond® PAG Film. The
following solutions are all known to shrink gels so thatthey tear off the support when reswollen.
1. Alcohol (methanol, ethanol, isopropanol) solutions of
45% or greater concentration.
2. DMSO (dimethylsulfoxide).
3. Perkin Elmer’ En3HANCE® autoradiographyenhancer. (Perkin Elmer also reports thatfluorography with EN3HANCE is seriously inhibitedby GelBond® PAG Film. The interference is more
pronounced with tritium.14 However, manycustomers have reported satisfactory results with35
S.)
Gel shrinkage by solution dehydration is analogous to
final gel drying. Inclusion of 5% glycerol preceding thedehydrating step has prevented disadhesion in many
instances. Dehydration by DMSO followed byrehydration to precipitate PPO is too severe, however,for the glycerol approach. No adequate preventive
measure is known for DMSO-caused disadhesion.

Related ProductsLong Ranger™ Solution
ProSieve™ 50 Solution
MDE™ Gel Solution®

GelBond PAG Film is manufactured for Lonza Rockland, Inc

High Degrees of Bisacrylamide Cross-Linking
Highly cross-linked 10% gels (19:1 acrylamide tobisacrylamide) have been reported to shrink severely
overnight in 25% isopropanol/10% acetic acid. If bonded
to GelBond® PAG Film, these gels curl up and crack
when dried, even after being soaked in as high at 20%
glycerol and carefully dried on a vacuum slab dryer.15

For Research Use Only. Not for use in Diagnostic
Procedures.

For more information contact Scientific Support at(800) 521-0390 or visit our website atwww.Lonza.com
Amersham, Pharmacia, Immobiline and Multiphor are trademarks ofAmersham Pharmacia Biotech. Bio-Rad and Protean are trademarksof Bio-Rad Laboratories. Cello-Seal is a trademark of Fisher Scientific
Co. Coomassie is a trademark of Imperial Chemicals Industries PLC.
3EN HANCE is a trademark of E.I. DuPont de Nemours & Co.
GeneAmp is a trademark of Hoffmann-LaRoche, Inc. NONIDET is atrademark of Shell Co. Life Technologies is a trademark of Life
Technologies, Inc. Scotch is a trademark of Minnesota Mining &Manufacturing Co. Triton is a trademark of Rohm & Haas Co.
Whatman is a trademark of Whatman Paper Ltd. Wratten, Kodak, andKodavue are trademarks of Eastman Kodak Co. GelBond is atrademark of FMC Corporation. All other trademarks herein are marksof the Lonza Group or its affiliates.
© 2010 Lonza Rockland, Inc.
All rights reserved.

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