PROTOCOL: Electrophoretic Separations of pancreatic enzyme samples:

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

(SDS-PAGE), Native-Polyacrylamide Gel Electrophoresis and
Isoelectric Focusing (IEF)

I. OBJECTIVE

The purpose of this experiment is to characterize pancreatic enzymes. Sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) will be used to
determine the molecular weight of the enzymes and isoelectric focusing (IEF) will be
used to determine the isoelectric point (pI) of the enzymes. Commercially
purchased authentic enzymes w ill be used as standards and Porcine

Pancreatic Enzyme Concentrate (PEC) and Pancreatic Enzyme Concentrate-High

Lipase (PEC-HL) will be used as enzyme samples.

II. INTRODUCTION

Pancreatic Enzyme Concentrate (PEC) and Pancreatic Enzyme Concentrate-High

Lipase (PEC-HL) are porcine pancreas derived drug substances. Electrophoretic
separations using polyacrylamide gel electrophoresis (PAGE) can be implemented to
separate specific proteins found in the pancreas such as amylase,
carboxypeptidase, elastase, lipase and proteases, trypsin and chymotrypsin.
Additionally, after electrophoretic separation, bands of protein can be further
analyzed and identified using antibodies and specific enzyme assays.

III. BACKGROUND

Polyacrylamide gel electrophoresis is one of the most frequently used techniques for the analysis
of proteins in a sample due to its sensitivity and ability to clearly and quickly resolve the
numerous protein constituents of a sample into bands. Gel electrophoresis typically involves
the migration of proteins through a gel matrix upon application of an electrical current. A sundry
of types of gel electrophoresis can be used depending on the type of analysis required. Native-
polyacrylamide gel electrophoresis (Native-PAGE) separates proteins primarily by their charge-
to-mass ratio and in their native conformations. This non-reducing and non-denaturing separation
technique maintains protein secondary and tertiary structure and allows for the detection of
biological activity and can improve detection by antibodies. Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) incorporates sodium dodecyl sulfate, an anionic
detergent, in sufficient excess to denature and fully saturate all proteins in a sample, giving them
a net negative charge and a uniform charge-to-mass ratio. -mercaptoethanol is used as a
reducing agent to reduce disulfide bonds and eliminate protein secondary structure. Since
proteins migrate through an SDS gel based primarily on their size, molecular weights for sample
proteins can be determined using molecular weight standards. Isoelectric focusing (IEF) uses
ampholytes (molecules that have both positively-charged and negatively-charged moieties) to
establish a pH gradient. Upon application of an electric field, proteins migrate to their isoelectric
point (pI) where they have no net charge and thus stop migration. Protein standards of known
isoelectric points are run concurrently with unknowns so that the pI of the unknown proteins can
be determined. After any type of electrophoretic separation, bands of protein can be visualized
with stains or submitted to further analyses such as activity staining, immunoblotting and mass
spectrometry.

Bio-Rad Precast Gel Systems provide a wide variety of polyacrylamide gels for
native, SDS and IEF experiments. Each batch is analyzed to ensure quality and
uniformity, which eliminates many of the pitfalls of hand-casting gels. The
Criterion precast gel system utilizes mid-size polyacrylamide gels and provides
high-resolution results with excellent reproducibility. Criterion Tris-HCl gels are
made without SDS in an array of acrylamide percentages so they can be used for
both SDS and native electrophoresis. Criterion IEF gels are available in a range of
pH gradients and contain no denaturing agents, which permits one-dimensional
separation under native conditions.

VI. EQUIPMENT

Bio-Rad Criterion Precast Gel System

Model # CRITERION Cell

Thermo Electron 2060P Power Supply

Belly Dancer Shaker

Bio-Rad GelAir Dryer

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Pipetman P20 Micropipet, 2-20 L

Pipetman P200 Micropipet, 20-200 L

Pipetman P1000 Micropipet, 100-1000 L

Beckman Microfuge 11

Gel Cutter Acrylic, Sigma (Cat. # G4778)

V. TEST SAMPLES
PEC or PEC-HL samples

Purchased amylase, carboxypeptidase, elastase, lipase and proteases,

trypsin and chymotrypsin.

VI. MATERIALS & REAGENTS

Bio-Rad Criterion Precast Polyacrylamide Gels (8.7 x 13.3 cm; 1.0 mm thick); 18
well; 30 L well capacity

Native (Cat. # 345-0033): 4-20% acrylamide (Tris-HCl), 2.6% bis-acrylamide

crosslinker

SDS (Cat. # 345-0033): 4-20% acrylamide (Tris-HCl), 2.6% bis-acrylamide

crosslinker

IEF (Cat. # 345-0072): pH 3-10, 5% acrylamide, 3.3% bis-acrylamide

crosslinker

10X Tris/Glycine Running Buffer, [concentration of 1X is 25 mM Tris, 192 mM glycine, pH 8.3]

10X Tris/Glycine/SDS Running Buffer, [concentration of 1X is 25 mM Tris, 192 mM glycine,
0.01% SDS, pH 8.3]
Native Sample Buffer, [62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% w/v bromophenol blue]
Precision Plus Protein Standards All Blue, Bio-Rad (Cat. # 161-0373)

Laemmli Sample Buffer, Bio-Rad (Cat. # 161-0737) [62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25%
glycerol, 0.01% w/v bromophenol blue]
-mercaptoethanol, electrophoresis grade, Sigma (Cat. # M7154)

Imperial Protein Stain, Pierce (Cat. # 24615)

IEF Standards, Broad Range pI 4.45-9.6, Bio-Rad (Cat. # 161-0310)

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10X IEF Cathode Buffer, Bio-Rad (Cat. # 161-0762) [concentration of 1X is 20 mM lysine, 20
mM arginine]
10X Anode Buffer, Bio-Rad (Cat. # 161-0761) [concentration of 1X is 7 mM phosphoric acid]
IEF Sample Buffer, [50% glycerol]

FisherBrand Sterile Gel Loading Tips, 1-200 L (Cat. # 02-707-81)

Nalgene Round Floating Microcentrifuge Tube Rack

Gel Drying Solution: 1X, Bio-Rad (Cat. # 161-0752) [contains water, ethanol]

GelAir Cellophane Support, Bio-Rad (Cat. # 165-1779)

VII. ELECTROPHORETIC SEPARATION: PROCEDURES

A. SDS-PAGE
1. Preparation of 1X Running Buffer: Add 45 mL 10X Tris/Glycine/SDS
Running Buffer to 405 mL distilled water. Mix gently but thoroughly.
2. Preparation of Sample Buffer: Under a fume hood, add 50 L -
mercaptoethanol to 950 L Laemmli Sample Buffer in a 2.0 mL
locking lid microcentrifuge tube. Vortex gently to mix.
3. Preparation of Samples: Accurately weigh out 20-25 mg of
pancreatin enzyme concentrate (PEC) or pancreatin enzyme
concentrate high lipase (PEC-HL) and transfer to a 2.0 mL
microcentrifuge tube. Add 1.0 mL distilled water and vortex
vigorously for five minutes. Centrifuge samples at 2000 x g (8000
rpm on Beckman Microfuge 11) for ten minutes. Dilute 50 L
sample with 100 L sample buffer. Bring approximately 200 mL
distilled water to a boil in a 600 mL beaker. Secure tubes in a
floating microcentrifuge tube rack and place in the boiling water
bath for 5 minutes. After five minutes, remove rack from bath and
allow samples to cool to room temperature.
4. Preparation of Criterion Precast Gel: Remove precast gel cassette
from storage container and rinse with a few mLs of distilled water.
Place cassette in one of the slots in the Criterion tank. Add
approximately 50 mL 1X Tris/Glycine/SDS Running Buffer to upper
buffer chamber. Remove well comb by gently pulling upward in a
uniform, concerted motion.
5. Loading of Samples: Using a micropipet with gel-loading tips, load
10-20 L of sample per well. Additionally, 10 L of the Bio Rad
Precision Protein Standards should be loaded into one or two wells.
6. Running Conditions: Once samples have been loaded, add
approximately 400 mL 1X Tris/Glycine/SDS Running Buffer to the
lower chamber of the cell (up to the FILL line). Snap the lid on the
chamber and plug the leads into the power source. Place the

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 chamber in cold room. Apply a constant voltage of 200 V for 55 min;
monitor and record the initial and final amperage.
7. Staining Protocol: After electrophoresis is complete, turn off the
power supply and disconnect the electrical leads. Remove the
Criterion cassette and pour off the buffer from the upper chamber.
Open the cassette by inverting it and cracking the welds using the
tool built into the lid of the tank. Transfer the gel to a Nalgene
storage container. Wash gel three times using 200 mL distilled
water for each wash. Each wash should last five min. Shake on an
orbital shaker at 55 rpm throughout each wash. Remove all water
from the staining container. Add approximately 100 mL Imperial
Protein Stain (enough to completely cover gel) and shake at 55 rpm
for two hours. Destain gel in 200 mL of distilled water while shaking
at 55 rpm. Place a folded KimWipe in the staining container during
the destain step to absorb excess stain and decrease the time
needed to fully destain the gel. Changing the distilled water and
KimWipe frequently will also decrease the time needed to obtain a
clear background. After destaining, dry gel according to gel drying
protocol that follows the separation protocols in this document.
B. Isoelectric Focusing
1. Preparation of 1X Cathode Buffer: Add 5 mL of IEF 10X Cathode
Buffer to 45 mL distilled water and mix thoroughly. Do not adjust
pH!
2. Preparation of 1X Anode Buffer: Add 40 mL with IEF 10X Anode
Buffer to 360 mL distilled water and mix thoroughly. Do not adjust
pH!
3. Preparation of Samples: Accurately weigh out 20-25 mg of
pancreatin enzyme concentrate (PEC) or pancreatin enzyme
concentrate high lipase (PEC-HL) and transfer to a 2.0 mL
microcentrifuge tube. Add 1.0 mL 50% glycerol and vortex
vigorously for five minutes. Centrifuge samples at 2000 x g (8,000
rpm on Beckman Microfuge 11) for ten minutes. Dilute 90 L
supernatant with 10 L IEF Sample Buffer (50% glycerol).
4. Preparation of Criterion Precast Gel: Remove precast gel from
storage container and rinse with a few squirts of distilled water.
Place cassette in one of the slots in the Criterion tank. Add
approximately 50 mL 1X Cathode Buffer to upper chamber. Remove
well comb by gently pulling upward in a uniform, concerted motion.
5. Loading of Samples: Using a micropipet with gel-loading tips, load
25 L of each sample per well. Additionally, 5.0 L of the IEF
Standards should be loaded into one well.
6. Running Conditions: Once samples have all been loaded, add
approximately 400 mL 1X Anode Buffer to the lower chamber of the
cell (up to the FILL line). Place the lid on the chamber and plug the
lid into the power source. Place the chamber in the cold room Apply
a constant voltage of 100 V for one to two hours. Then, increase the
voltage to 250 V for one hour. Finally, increase the voltage to 500 V
for 30 minutes. Monitor and record the initial and final amperages
for each voltage change.

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 7. Staining Protocol: After electrophoresis is complete, turn off the
power supply and disconnect the electrical leads. Remove the
Criterion cassette and pour off the buffer from the upper chamber.
Open the cassette by inverting it and cracking the welds using the
tool built into the lid of the tank. Transfer the gel to a Nalgene
staining container. Add approximately 100 mL (just enough to cover
the gel) of 20% trichloroacetic acid (TCA) to fix the gel. Shake at 55
rpm for one hour. Rinse with disrilled water and add approximately
100 mL Imperial Protein Stain (enough to completely cover gel)
and shake at 55 rpm for two hours. Destain gel in 200 mL of
distilled water while shaking at 55 rpm. Place a folded KimWipe in
the staining container during the destain step to absorb excess
stain and decrease the time needed to fully destain the gel.
Changing the distilled water and KimWipe frequently will also
decrease the time needed to obtain a clear background. After
destaining, dry gel according to gel drying protocol that follows the
separation protocols in this document.
C. Native-PAGE
1. Preparation of 1X Running Buffer: Add 100 mL 10X Tris/Glycine
Running Buffer to 900 mL distilled water. Mix thoroughly.
2. Preparation of Samples: Accurately weigh out 20-25 mg of
pancreatin enzyme concentrate (PEC) or pancreatin enzyme
concentrate high lipase (PEC-HL) and transfer to a 2.0 mL
microcentrifuge tube. Add 1.0 mL distilled water and vortex
vigorously for five minutes. Centrifuge samples at 2000 x g (8000
rpm on Beckman Microfuge 11) for ten minutes. Dilute 50 L
supernatant with 100 L Native Sample Buffer.
3. Preparation of Criterion Precast Gel: Remove precast gel from
storage container and rinse with a few squirts of distilled water.
Place cassette in one of the slots in the Criterion tank. Add
approximately 40 mL 1X Tris/Glycine Running Buffer to upper
chamber. Gently remove well comb by pulling upward in a uniform
motion.
4. Loading of Samples: Using a micropipet with gel-loading tips, load
20 L of each sample per well.
5. Running Conditions: Once samples have been loaded, add
approximately 400 mL 1X Tris/Glycine Running Buffer to the lower
chamber of the cell (up to the FILL line). Snap the lid on the
chamber and plug the leads into the power source. Place the
chamber in the cold room. Apply a constant voltage of 125 V for
120 minutes; monitor and record the initial and final currents.
6. Staining Protocol: After electrophoresis is complete, turn off the
power supply and disconnect the electrical leads. Remove the
Criterion cassette and pour off the buffer from the upper chamber.
Open the cassette by inverting it and cracking the welds using the
tool built into the lid of the tank. Transfer the gel to a Nalgene
staining container. Wash gel once for five min using 200 mL distilled
water. Shake on an orbital shaker at 55 rpm throughout wash.
Remove all water from the staining container. Add approximately

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 100 mL Imperial Protein Stain (enough to completely cover gel)
and shake at 55 rpm for one hour. Rinse gel in 200 mL of distilled
water while shaking at 55 rpm. Place a folded KimWipe in the
staining container during the destain step to absorb excess stain
and decrease the time needed to fully destain the gel. After
destaining, dry gel according to gel drying protocol that follows the
separation protocols in this document.
D. Drying of Acrylamide Gels (SDS, Native and IEF)
1. Gel Preparation: Equilibrate gel in Gel Drying Solution for 30
minutes while shaking at 55 rpm. Use the Gel Cutter to slice off the
thicker edge along the bottom of the gel (except for IEF gels).
2. Frame Assembly: Place the molded bottom frame on the assembly
table with the textured side up. Submerge a sheet of cellophane
and center the wetted sheet on top of the assembly table. Smooth
out any wrinkles or bubbles. Place gel(s) on cellophane sheet. Use a
wash bottle to add distilled water around the edges of the gel and in
the wells. Wet a second sheet of cellophane and lay this sheet over
the top of the gel(s). If air bubbles are trapped under the sheet,
raise the cellophane and lower it again using a wash bottle to squirt
more distilled water across and around the gel. Smooth away any
remaining bubbles and place the stainless steel top frame over the
cellophane and center it over the bottom frame. Clamp the top
frame to the bottom frame using two clamps per side. Lift the
clamped drying frame off the assembly table and slide it into one of
the shelves in the dryer.
3. Drying Conditions: Dry gels for 70 min with heat. After this time,
feel the entire gel with the back of your hand. If it feels cold or
moist, place back in the dryer and dry with heat for 5 additional
min. Repeat, if necessary. Once gel has dried and cooled to room
temperature, unhook the clamps and cut away any superfluous
cellophane from around the gel. Cellophane can be trimmed right
up to the edge of the gel. Secure dried gel in appropriate laboratory
notebook using photo-mounting corners.