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Document # 18116-0311-8
Rockland, ME 04841 USAProSieve™ 50 Gel Solution

Introduction

Precautions

ProSieve™ 50 Gel Solution is a unique, modified acrylamide
formulation developed for high performance protein gel
electrophoresis. Gels prepared from this solution have several
advantages over conventional polyacrylamide gels.
Specifically, when cast at a single concentration and used witha Tris/Tricine electrode buffer, ProSieve™ 50 Gels willseparate proteins as gradient polyacrylamide gels (see Tablebelow). Alternately, 8% ProSieve™ 50 Gels, when used with aTris/Glycine electrode buffer, will easily separate proteinslarger than 200 kDa.

Wear gloves and use all safety precautions routinely used
when handling acrylamide solutions. Material
Safety Data Sheets (MSDS) for this product are available bycontacting our Customer Service Department at
1-800-638-8174 or on our website at www.lonza.com.

Reagent Preparation
Electrode Buffers
10X Tris/Tricine/SDS Electrode Buffer(Dilute to 1X with deionized water prior to use):Tris Base
121.0 gTricine179.0 g
10% SDS solution*100 ml
Deionized Water to1000 ml
Do not adjust the pH.
Store at room temperature.
*Omit for native proteins.

ProSieve™ 50 Gels have a uniform pore size distribution
allowing for faster protein mobility than in conventional
polyacrylamide gels. These gels are also stronger, less likely totear, and easier to handle.

Table 1: Gel Percentage Guidelines for Gradient Gel
Separation Performance in Tris/Tricine Electrode
BufferProtein Size
Separation Range
Optimal Size Range(kDa)Resolvable Size
Range (kDa)

8%

10%

12%

14%

25-15015-10010-70

5-50

15-300

5-200

5-150

2-100

10X Tris/Glycine/SDS Electrode Buffer(Dilute to 1X with deionized water prior to use)
Tris Base
30.0 gGlycine144.0 g10% SDS solution*
100 mlDeionized Water to1000 ml
Do not adjust the pH.
Store at room temperature.

Materials*Omit for native proteins.

50% ProSieve™ 50 Gel Solution

2X Denaturing Sample Buffer
Storage

0.5 M Tris-HCI, pH 6.8
2.5 mlGlycerol
2.0 ml
10% SDS solution*
4.0 ml
0.1% (w/v) Bromophenol Blue
0.5 ml2-mercaptoethanol*
0.5 mlDeionized Water to10 mlStore at -20°C.
*Omit for native proteins.
Caution: 2X Sample Buffer containing 2-mercaptoethanol
should be prepared in a fume hood.
0.2 M (final concentration) Dithiothreitol (DTT) may be used inplace of 2-mercaptoethanol.

ProSieve™ 50 Gel Solution should be stored at room temperature.
It is stable for 24 months from the date of manufacture.

Reagents and solutions not provided•
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1.5 M Tris-HCI, pH 8.8
1 M Tris-HCI, pH 6.8
10% (w/v) SDS solution
0.1% (w/v) Bromophenol BlueAmmonium PersulfateTEMED (N,N,N´,N´-tetramethylenediamine)
Deionized Water
Tris/Tricine Electrode Buffer or
Tris/Glycine Electrode Buffer1Gel preparation and pouring*APS solutions should be prepared fresh just prior to use.
‡Omit for native protein analysis.

Table 1: Preparation of a 10 ml Resolving Gel(Proportionally adjust volumes for larger gels.)Components

8%Gel

10% Gel

12% Gel

14% GelDeionized Water
ProSieve™ 50
Gel Solution

5.7 ml
1.6 ml5.3 ml2.0 ml

4.9 ml2.4 ml

4.5 ml
2.8 ml

1.5M Tris-HCI,pH 8.8

2.5 ml

2.5 ml

2.5 ml

2.5 ml

10% SDS
Solution‡

0.1 ml0.1 ml0.1 ml0.1 ml

10% APS*
100 µl100 µlTEMED
4.0 µl4.0 µlAPS solutions should be prepared fresh just prior to use.
*100 µl
4.0 µl100 µl4.0 µl8.

Sample Preparation
Combine equal volumes of your protein sample with the 2X
sample buffer in a microcentrifuge tube and place the tube into
a boiling water bath for 2 to 3 minutes. After heating, briefly
centrifuge the tube to collect droplets. For native proteins, donot apply heat.
1. Load your samples immediately or place them on ice until
ready for use. Treated samples can be stored at -20°C.
NOTE: The amount of sample to load on the gel depends
on sample composition (e.g., complex protein mixtures orpolypeptides) and the detection method. WhenCoomassie® Blue staining, load 1 µg-10 µg of a highly
purified protein per lane or load 25 µg-50 µg of a complex
mixture (cell lysates) per lane. When silver staining, load
10- to 100- fold less protein per lane.

‡Omit for native protein analysis.

1.

Clean your glass plates and wipe them with ethanol.
Allow them to air-dry.
2. Assemble the gel cassette according to manufacturer’s
instructions or use preassembled cassettes.
3. Prepare the resolving gel. Place the specified quantitiesof the first five components from Table 1 in a clean
beaker. Thoroughly mix the components.
NOTE: The ProSieve™ 50 Gel Solution does not require
degassing. If required by your procedure, degas the solution
prior to the addition of SDS.
4. Add the specified amount of TEMED and mix the solution
by inversion or gentle swirling.
5. Pour the gel solution between the cassette glass plates.
Carefully layer water or water-saturated n-butanol along
the surface of the gel solution with a pipette. Allow the gel
to polymerize for at least 30 minutes at room temperature
before casting the stacking gel.
NOTE: Resolving gels can be stored for up to one weekat 4°C. To prepare the gel for storage, decant the overlay
and thoroughly rinse the top of the gel with 1X electrodebuffer. Keep the gels moist by layering a small volume of
1X electrode buffer on top of the gel and wrapping the gelin plastic wrap.
6. Decant the water or n-butanol prior to casting the stacking
gel. Thoroughly rinse the top of the gel with 1X electrodebuffer and decant. (If n-butanol was used as an overlay,
several washings will be required.) Make sure that all of
the overlay is removed completely by aspirating or byabsorbing excess fluid with a paper towel or blotting
paper.
7. Prepare the stacking gel solution by combining thespecified quantities from Table 2 into a clean beaker.
Thoroughly mix the components.

Electrophoresis
1.

2.
3.
4.

5.

6.

7.

1 ml
750 µl100 µl130 µl
10 µl
10 µl
1 µl

Prepare enough working solution of 1X electrode buffer tocompletely fill the upper and lower buffer chambers of yourelectrophoresis apparatus.
Remove the comb from the stacking gel and thoroughly
flush the wells with 1X electrode buffer.
Mount the gel cassette on the electrophoresis apparatus
according to the manufacturer's instructions.
Fill the upper and lower buffer chambers with 1X electrodebuffer. Flush the sample wells several times and fill withelectrode buffer.
Load equal volumes of the samples on the gel (ifpossible). Load empty wells with an equal volume of 1X
sample buffer. If lanes are left empty or loaded withunequal volumes, adjoining samples may spread during
electrophoresis.
Electrophorese samples at 130 V (constant voltage) for
minigels (10 cm x 10 cm x 0.1 cm) or at approximately 200
volts for larger gels (18 cm x 16 cm x 0.1 cm).
Stop the electrophoresis once the tracking dye reaches
the bottom of the resolving gel. Approximate times forelectrophoresis are:
Minigels
Large Gels

Table 2: Preparation of a 5% Stacking Gel
Components
Deionized Water
ProSieve™ 50 Gel Solution1M Tris-HCI, pH 6.8
10% SDS Solution‡
10% APS*TEMEDPour the stacking gel solution on top of the resolving gelwith a pipette. Insert the comb and add sufficient stacking
gel solution to completely fill the cassette. Allow thestacking gel to polymerize for 30 to 40 minutes at room
temperature.
NOTE: Stacking gels should be prepared fresh prior touse. If necessary, polymerized gels can be stored with thecomb in place and the gel wrapped with plastic wrap at
4°C for up to 16 hours with no loss in performance.

5 ml
3.75 ml
0.5 ml
0.65 ml
50 µl50 µl
5.0 µl8.

9.

2

60 minutes
90 minutes(Tris/Tricine electrode buffer)
Remove the protein gel cassette from the apparatus and discard
the electrode buffer. Carefully separate the two plates while
maintaining gel orientation. Trim one corner of the gel so sample
order is not lost during drying or staining.
Remove the gel from the plate and proceed with protein
detection or transfer.

Protein Detection
Conventional protein detection techniques used forpolyacrylamide gels are compatible with gels prepared from theProSieve™ 50 Gel Solution.

Approximate Detection Limits
StainSensitivityCoomassie®
Brilliant Blue0.3 µg-1.0 µg
per protein band

Silver Stain
2.0 µg -5.0 µg
per protein band

Blotting and Drying
Conventional techniques for blotting, drying, and preserving
polyacrylamide gels are compatible with ProSieve™ 50 Gels.

Ordering Information
Catalog No.
50617
50618

Description
ProSieve™ 50 Gel Solution
ProSieve™ 50 Gel SolutionSize
125 ml
250 mlRelated Products
PAGEr™ Precast Gels
ProSieve™ Color Protein Markers
ProSieve™ Protein Markers
SYPRO® Red Gel Stain
SYPRO® Orange Gel Stain
SYPRO® Tangerine Gel Stain
AccuGENE™ Tris-Glycine BufferAccuGENE™ Tris-Glycine SDS Buffer
ProSieve™ 50 Gel Solution is intended For Research UseOnly.

For more information contact Scientific Support at(800) 521-0390 or visit our website at www.lonza.com.

SYPRO is a trademark of Molecular Probes, Inc., Coomassie
is a registered trademark of ICI Lourdes. All other trademarks
herein are marks of the Lonza Group or its affiliates.

© 2011 Lonza Rockland, Inc.
All rights reserved.

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