PROTOCOL:
I.
Electrophoretic Separations of pancreatic enzyme samples: Sodium
Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE),
Native-Polyacrylamide Gel Electrophoresis and Isoelectric Focusing (IEF)
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 will 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 (PECHL) 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. Nativepolyacrylamide gel electrophoresis (Native-PAGE) separates proteins primarily by their chargeto-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 sulfatepolyacrylamide 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
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]
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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)
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 chamber in cold room. Apply a constant voltage of
200 V for 55 min; monitor and record the initial and final amperage.
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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 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.
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
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(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 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.
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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.
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