Nippon Genetics Europe FastGene PG01 User manual

NIPPON Genecs EUROPE GmbH Mariaweilerstraße 28-30, 52349 Düren, Germany,
Tel: [phone], Fax: [phone], E-Mail: info@nippongenecs.de, www.nippongenecs.eu
PAGE PROTEIN SYSTEM
PG01
Manual

NIPPON Genecs EUROPE
FastGene® PAGE Protein System

NIPPON Genecs EUROPE
FastGene® PAGE Protein System
CONTENT
P. 5
P. 7
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P. 11
P. 13
P. 13
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P. 16
P. 19
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P. 31
P. 31
1. INTRODUCTION..................................................................................................
2. SYSTEM COMPONENTS....................................................................................
2.1 Ordering Informaon............................................................................
2.2 FastGene®PAGE Protein Sytem (PG01)..........................................
2.2.1 PAGE System Set Components.........................................
2.2.2 Gel Hand-Casng Set Components.................................
3. SYSTEM OPERATION..........................................................................................
3.1 Gel Hand-Casng..................................................................................
3.1.1 Assembly of Gel Hand-Casng Set..................................
3.1.2 Polyacrylamide Gel casng................................................
3.2 PAGE System Operaon......................................................................
3.2.1 U Sealing Strips for Pre-Cast and Hand-cast gels.........
3.2.2 Gel Cassee Assembly........................................................
3.2.3 PAGE System Chamber Assembly....................................
3.2.4 Sample Preparaon..............................................................
3.2.5 Sample Loading.....................................................................
3.2.6 Performing Electrophoresis................................................
4. TROUBLE SHOOTING........................................................................................
5. WARRANTY...........................................................................................................
6. CONTACT INFORMATION................................................................................

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FastGene® PAGE Protein System

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FastGene® PAGE Protein System 5
Thank you for purchasing the FastGene® PAGE Protein System. The system is compatible
with hand-cast gels and pre-cast gels (e.g. FastGene®gels, Bio-Rad TGX™gels and
ThermoFisherTM mini gels) for electrophoretic protein separation. It can run a maximum of
4 gels simultanously.
PAGE stands for Polyacrylamide Gel Electrophoresis and describes an analytical method
in biochemistry for the separation of differently sized protein mixtures in an electric field.
During PAGE, proteins migrate through a gel matrix in response to an applied electric
field. Smaller proteins travel faster through the gel than larger proteins, leading to a size
dependent separation.
The most common form of PAGE is SDS-PAGE. The detergent sodium dodecyl sulfate
(SDS) is added for PAGE sample preparation and is also part of the buffer composition.
The treatment with the harsh detergent leads to full protein denaturation and unfolding.
SDS binds to hydrophobic parts of the unfolded protein and masks the intrinsic charge
of the protein with its own negative charge. As a consequence, SDS-protein complexes
migration is predominantly dependent on the size of the protein, allowing an estimation
of its molecular weight.
Aer the PAGE protein separaon is completed, protein visualizaon and detecon is
carried out. Protein visualizaon is achieved by the use of protein specic stains. Aer
staining, images can be taken of the gel for analycal purposes.
1. INTRODUCTION

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2.1 Ordering Information
Following sets / components are available, addionally to the FastGene® PAGE Protein
System (PG01):
Cat. No. Product Content
PG01 FastGene®PAGE Protein System Complete protein PAGE set
(for content list see table on next page)
PG02 FastGene®Comb Set 075 Gel hand-casng set (for 0.75 mm gel thickness)
(for content list see table on next page)
PG03 FastGene®Comb Set 150 Gel hand-casng set (for 1.5 mm gel thickness)
(for content list see table on next page)
PG04 FastGene® Glass plates short Short at glass plates for hand-cast gels
(10 glass plates)
PG05 FastGene®Gel/Blot chamber tank Large gel electrophoresis buer tank
(1 chamber tank)
PG06 FastGene® Gel casng clip Frame for holding hand-cast gels (4 pieces)
PG07 FastGene®Sealing gaskets Sealing gaskets for gel casng (5 pieces)
2. SYSTEM COMPONENTS

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2.2 FastGene®PAGE Protein System (PG01)
Please check the system and the packing list when you rst open the packing case of
the FastGene® PAGE Protein System. If you nd there is something wrong with the
system or the packaging content, please contact NIPPON Genecs EUROPE or your local
distributor.
The FastGene® PAGE Protein System (PG01) contains PAGE System components and Gel
casng set components for hand cast gels:
FastGene®PAGE Protein System (PG01) Qty
PAGE System set 1 set
Gel hand-casng set (1 mm gel thickness) 1 set
FastGene®PAGE Protein System (PG01) - PAGE System Qty
PAGE System
Inner electrophoresis chamber with electrodes
(with side clip for gel holder 2x)
1
Inner electrophoresis chamber without electrodes
(with side clip for gel holder 2x)
1
U sealing strip long for 10 x 10 cm gels
(e.g. ThermoFisher mini gels)
4
U sealing strip short for Bio-Rad TGX™ gels
(10 x 8 cm)
4
U sealing strip FastGene (10 x 8 cm) 4
Plasc dummy cassee short (10 x 8 cm) 1
Plasc dummy cassee long 1
FastGene®Gel/Blot chamber lid with electrodes (and power cable) 1
FastGene®Gel/Blot chamber tank (PG05) 1
Gel shovel 5
2.2.1 PAGE System Set Components
The PAGE System set components of PG01 allow to execute electrophorec separaon
of proteins with the use of polyacrylamide gels.

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3. Inner electrophoresis chamber
with electrodes (with side clip for
gel holder 2x)
4. Inner electrophoresis chamber
without electrodes (with side clip
for gel holder 2x)
6. U sealing strip5. Hand-cast gel /
Pre-cast gel /
Plasc dummy cassee
1. FastGene®Gel/Blot chamber lid
with electrodes (and power cable)
2. FastGene®Gel/Blot chamber tank
Fig. 1: PAGE system components

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Descripon of PAGE system components (depicted in Fig. 1):
1. FastGene®Gel/Blot chamber lid with electrodes (and power cable):
The lid is posioned on top of the inner electrophoresis chambers and the FastGene®
Gel/Blot chamber tank. The power cables (cathode and anode) are connected to the
power supply.
2. FastGene®Gel/Blot chamber tank:
The chamber tank holds both inner electrophoresis chambers with a maximum of
four gels/plasc dummy cassees. The chamber is lled with running buer during
the electrophoresis run.
3. Inner electrophoresis chamber with electrodes:
Holds two gels/plasc dummy cassees via the two gel side clip holders. The
inner chamber is lled with running buer during the electrophoresis run. The lid is
connected to the inner chamber electrodes.
4. Inner electrophoresis chamber without electrodes:
Holds two gels/plasc dummy cassees via the two gel side clip holders. The inner
chamber is lled with running buer during the electrophoresis run. The lid is placed
on top of the inner chamber electrodes.
5. Hand-cast gel / Pre-cast gel / Plasc dummy cassee:
The system is compatible with hand-cast gels and pre-cast gels (e.g. FastGene®
gels, Bio-Rad TGX™gels and ThermoFisherTM mini gels) for electrophoretic protein
separation. In case 1 or 3 gels are used with the system, a gel is replaced with a
short or long plastic dummy cassette.
6. U sealing strip:
The U sealing strips are placed between the inner electrophoresis chambers and
the gels/plastic dummy cassettes on both sides. They seal the inner electrophoresis
chamber (cathode) from the (outer) gel/blot chamber tank (anode). Depending on
the gel size and gel type, different U sealing strips are used (See page 19 for the use
of compatible U sealing strips).

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2.2.2 Gel Hand-Casting Set Components
The FastGene® PAGE Protein System (PG01) is equipped with a gel hand-casng set
for the preparaon of hand-cast (polyacrylamide) gels with 1 mm gel thickness. The
FastGene®Comb Set 075 (PG02) and FastGene®Comb Set 150 (PG03) are available for
the preparaon of 0.75 mm gels and 1.5 mm gels, respecvely.
FastGene® PAGE Protein System (PG01) - Gel hand-casng set Qty
Gel hand-casng set (1 mm gel thickness)
Comb 1 mm 10 wells
PG02 (0.75 mm) PG03 (1.5 mm)
5
Comb 1 mm 15 wells 5
Glass spacer long 1 mm 5
Glass plates short (PG04) 10
Gel casng base 4
Gel casng clip (PG06) 4
Sealing gaskets (PG07) 5
Glass plate holder 1
Tube holder 1
Glass plate short
Glass spacer long 1 mm
(0.75 mm, 1.5 mm)
Gel casng clip
Gel casng base
Sealing gasket
Fig. 2: Gel hand-casng set components

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3.1.1 Assembly of Gel Hand-Casng Set
Assemble the glass spacer (1 mm, 0.75 mm or 1.5 mm) together with a glass plate
on top.
Glass plate short Glass spacer long 1 mm
(0.75 mm, 1.5 mm)
1
3. SYSTEM OPERATION
Please read the instrucons manual carefully and operate the system accordingly.
3.1 Gel Hand-Casting
If you are using ready-to-use pre-cast gels, skip this secon and connue with secon
3.2 PAGE System Operaon.
Place the gel casng clip on a horizontal lab desk and keep both hinges of the frame
open. Posion the short glass plate towards the front and slide the assembled
glass spacer and glass plate into the gel casng clip from the top.
2

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The two glass plates should be aligned and both rest on the two bulges at the
boom of the gel casng clip. Close the hinges on both sides of the gel casng clip.
Bulges
Gel casng clip
(hinges closed)
Glass plate short
(in front) Glass spacer long 1 mm
(0.75 mm, 1.5 mm)
Gel casng clip
(hinges open)
Glass plate short
(in front)
Glass spacer long 1 mm
(0.75 mm, 1.5 mm)
Bulges
3
Carefully check that both glass plates are level with each other and are aligned at
the boom of the gel casng clip. Adjust if necessary. A wrong alignment can lead
to leakage during gel casng.
4

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Put a sealing gasket in the rectangular cavity in the front of the gel casng base.
Posion the closed gel casng clip with glass plates on top of the sealing gasket,
as shown below.
Gel casng clip
with glass plates
(hinges closed)
Sealing gasket
Gel casng base
Fix the the glass plates and gel casng clip to the gel casng base by pulling
the spring leverage to the front and securing the top of the glass spacer (1 mm,
0.75 mm, 1.5 mm)
Spring leverage
Connue with polyacrylamide gel casng. Repeat steps 1-6 when casng more
than one gel.
5
6
7

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3.1.2 Polyacrylamide Gel Casng
Soluon
Components
Stacking Gel Resolving Gel Final
concentraon
4 % 12 % X % (for 15 ml only)
30 % Acrylamide/Bis 0.8 ml 6 ml (0.5*X) ml variable
0.5 M Tris HCl, pH 6.8 1.5 ml ------ ------ 0,125 M
1.5 M Tris HCl, pH 8.8 ------ 3.75 ml 3.75 ml 0,375 M
10 % SDS 60 µl 150 µl 150 µl 0.1 %
H20 (deionized) 3.604 ml 5.01 ml 11,01 - (0.5*X) ml ------
TEMED 6 µl 15 µl 15 µl 0,1 %
10 % APS 30 µl 75 µl 75 µl 0,05 %
Total Volume 6 ml 15 ml 15 ml
For ideal protein separaon, hand-cast polyacrylamide gels are divided into two parts,
a stacking gel and a resolving gel. The stacking gel has a lower pH (pH 6.8) and a lower
polyacrylaymide percentage (4%) than the resolving gel. It is cast around the comb on
the upper part of the gel. The purpose of the stacking gel is to line up the loaded protein
samples so they can enter the resolving gel simultanously. The resolving gel has a higher
pH (pH 8.8) and a higher gel percentage (~7.5 % - ~20 %) than the stacking gel. It separates
proteins based on their molecular weight.
For preparing the stacking gel and resolving gel, mix the gel soluons listed in the
following table without TEMED and APS. The component volumes are given for a
total volume of 6 ml (4 %) stacking gel and 15 ml (12 %, X %) resolving gel. See the
table on page 17 for required volume per gel.
1
Stacking Gel
Resolving Gel
Protein migraon
Protein bands
}*
*Add later,
right before gel casng
(step 5 and step 7)
Fig. 3: Stacking gel and resolving gel

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The following table shows the gel volumes of 0.75 mm, 1 mm and 1.5 mm gel
casng plates. If necessary, adjust the total volumes for used gel thickness and
number of hand-cast gels accordingly.
2
3
4
Hand casng gel
thickness
Gel volume
(stacking gel + resolving gel)
0.75 mm ~ 6.06 ml
1 mm ~ 4.54 ml
1.5 mm ~ 9.09 ml
Degas the polyacrylamide gel soluon without TEMED and APS and prepare the
hand gel casng set in the meanme (see 3.1.1 Assembly of Gel Hand-Casng set).
Insert a comb between the two glass plates of the assembled gel casng set to
mark the casng level of the resolving gel and stacking gel. A sucient amount of
stacking gel is needed for proper protein band sharpness. Mark ~1 cm below the
teeth of the comb on the glass plate. The resolving gel will be poured up to this
level and covered with stacking gel aerwards. Remove the comb again.
Glass plate short
(in front)
Glass spacer long 1 mm
(0.75 mm, 1.5 mm)
Comb (e.g. 1 mm, 15 well)
Mark: ~ 1 cm below
teeth of comb

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5Resolving gel:Add TEMED and APS to the degassed polyacrylamide resolving gel
soluon. Gently swirl the vessel without drawing air bubbles into the soluon. This
will start the polymerizaon reacon. Subsequently pour the resolving gel soluon
between the glass plates using a glass pipee, unl the mark on the glass plate
(from step 4) is reached. Avoid air bubbles during pouring.
9
6
7
8
Immediately cover the resolving gel soluon with 100% isopropyl alcohol (propan-
2-ol) or water saturated n-butanol. Let the resolving gel soluon polymerize for
45 - 60 min. Aerwards, pour o the solvent and rinse the top of the resolving gel
with deionized H2O.
Stacking gel: For pouring the stacking gel, rst dry the top of the resolving gel by
using lter paper.
Add TEMED and APS to the degassed polyacrylamide stacking gel soluon. Gently
swirl the vessel without drawing air bubbles into the soluon. Pour the stacking
gel soluon between the glass plates on top of the resolving gel using a glass
pipee, unl the brim of the front glass plate is reached. Avoid air bubbles during
pouring. Carefully and slowly insert the comb to avoid air trapping under the teeth
of the comb. Insert the comb completely and align with the glass plate.
Let the stacking gel soluon polymerize for 30 - 45 min. If the gel is subsequently
used, carefully remove the comb by gently pulling it out and rinse the wells with
H2O or buer. If the gel is used at a later me, keep comb inserted and store the
gel cassee in a sealed plasc bag at 4 °C.
Further advice for hand gel casng
• Clean glass plates thoroughly with ethanol before using and avoid detergent
residues.
• For opmal results, only use high quality reagents. Always prepare fresh APS
soluons and avoid using TEMED soluons that are older than three months.
• Do not store gels for a long period of me. Do not freeze the gels.

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3.2 PAGE System Operation
3.2.1 U Sealing Strips for Pre-Cast and Hand-Cast Gels
Ready-to-use pre-cast gels oer easier and faster handling, increased quality control and
the possibility of storing gels for a longer period of me. The FastGene® PAGE Protein
System is compatible with Pre-cast gels from different leading manufacturers (e.g.
FastGene®gels, Bio-Rad TGX™ gels and ThermoFisherTM mini gels).
Compatibility of different pre-cast gels is assured by matching U sealing strips, which
are placed as a sealing between the inner electrophoresis chambers (with / without
electrodes) and the gels on both sides.
The FastGene® PAGE Protein System comes with three different U sealing strips to ensure
maximum compatibility (see Figure 3). The short U sealing strip without a notch is used
with 10 x 8 cm FastGene®Pre-cast gels. The short U sealing strip that contains a notch
in the upper area of the seal is used with 10 x 8 cm Bio-Rad TGXTM gels and hand-cast
gels. The long U sealing strip long contains additional sealing and is used with 10 x 10 cm
ThermoFisherTM mini gels.
Fig. 4: U sealing strips compability with Pre-cast gels and Hand-cast gels
U sealing strip FastGene®
• 10 x 8 cm gels
• FastGene®Pre-cast gels
U sealing strip short
• 10 x 8 cm gels
• Bio-Rad TGXTM
• Hand-cast gels
U sealing strip long
• 10 x 10 cm gels
• ThermoFisherTM mini
U sealing strip FastGene®(10 x 8 cm) FastGene®Pre-cast gels
U sealing strip short for Bio-Rad TGX™ gels (10 x 8 cm) Bio.Rad TGXTM, Hand-cast gels
U sealing strip long for 10 x 10 cm gels ThermoFisherTM mini gels
addional sealingnotchno notch

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3.2.2 Gel Cassette Assembly
If two gels are used (or one gel and one Plasc dummy cassee), the gels should be placed
into the inner electrophoresis chamber with electrodes.
If four gels are used (or three gels and one Plasc dummy cassee), the inner
electrophoresis chamber without electrodes should be used addionally. Do not use the
inner electrophoresis chamber without electrodes only for running one or two gels.
Aer inserng the compable U sealing strip into the Inner electrophoresis
chamber(s) (with / without electrodes), place the electrophoresis core onto a clean
and at desk.
1
Remove the combs from the gels and place the pre-cast gels / hand-cast gels /
plasc dummy cassee with the short glass / sample wells facing towards the
inside of the Inner electrophoresis chamber. Rest the lower part of a gel cassee
on the two steps posioned at the boom part of the inner electrophoresis
chamber and move the upper part of the gel cassee towards the U sealing strip.
2