Product Name:

DynaMarker®

Code No:

DM670L

Lot No:002CA04

Size:

200 μl ×3 (DM670 ×3)

Storage:

4 °C

Stability:

12 months at 4 °C

Storage Buffer:

50 mM Tris-HCl (pH6.8), Urea, SDS, Glycerol, EDTA

Protein MultiColor Stable, Low Range, Large(120 mini-gel lanes)

Description
DynaMarker®

Protein MultiColor Stable, Low Range is a pre-stained protein molecular weight marker. Themarker is supplied in gel loading buffer for direct loading onto SDS-PAGE without heating or adding
reducing agents. It is easy to start electrophoresis since the marker can be stored and stable at 4 °C in aliquid state. The marker consists of seven prestained protein standards, Blue, Red, Green and Purple,ranging in appropriate molecular weight from 2 kDa to 46 kDa. The marker is suitable for monitoring gelelectrophoresis and electrophoretic transfer onto membranes. The protein concentrations are optimized togive uniform band intensities.

－ Ovalbumin－ Carbonic Anhydrase－ Trypsin Inhibitor－ Lysozyme－ Aprotinin－ Insulin, B chain－ Bacitracin

Electrophoresis profile of DynaMarker Protein MultiColor
Stable, Low Range (5 μl) on 16% polyacrylamide (3 % C)
Gel / Tris-Tricine-SDS as running buffer.

Protocol1.

Take the marker out of refrigerator.

2.

Load 5 μl for mini-gels or more for large size gels.

3.

Load your samples.

4.

Start electrophoresis.

Note: There is no need to heat or add reducing agents.

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BioDynamics Laboratory Inc.
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ContentsProtein

Color

Apparent molecular weight (kDa) *Ovalbumin

Blue

46.3

Carbonic Anhydrase

Red30.8

Trypsin Inhibitor

Blue22.6

LysozymeBlue

17.0

Aprotinin

Green

8.7

Insline, B chain

Blue

3.9

Bacitracin

Purple1.7
Running buffer : Tris-Tricine-SDS buffer (1)Note: As covalently bound dye affects protein mobility, each batch of our prestained protein marker is calibrated
against unstained standards. A prestained protein marker should be used for approximate molecular weightdetermination. For precise molecular weight determination, you should use an unstained molecular weight marker.
* : The apparent molecular weight values are lot specific.

Recommended usageDynaMarker®

Protein MultiColor Stable, Low Range is suitable for monitoring low molecular weight protein

on polyacrylamide gel electrophoresis using Tris-Tricine-SDS as running buffer (1), and for monitoring
electrophoretic transfer.
One example is shown below:
・Detection of β-Amyloid (1-42)
1) Electrophoresis
1-1) Preparation of reagents (2)・Acrylamide-Bis stock solution (T:49.5%, C:3% )Acrylamide

48 g

Bis-acrylamide

1.5 g

dH2O

to 100 mL

・3× Gel buffer
3 M Tris-HCl (pH8.45), 0.3 % SDS
・10× Anode buffer
1.0 M Tris-HCl (pH8.9)
・10×Cathode buffer
1.0 M Tris, 1.0 M Tricine, 1.0 M SDS (pH adjustment is not necessary)

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・4×Sample buffer
150 mM Tris-HCl (pH7.0), 12 % SDS, 6 % mercaptoethanol, 30 % Glycerol, 0.05 % CBB1-2) Preparation of 16 % polyacrylamide (3 % C) Gel
Prepare the 16 % separating gel solution according to the following table and pour the gel solution into
the mold of a vertical gel apparatus. The separating gel is overlaid with several drops of water and left
until polymerized adequately, and then the overlaid water was replaced by a 4 % stacking gel.
4 % stacking gel

16 % separating gel

Acrylamid-Bis stock solution

1 ml

10 ml

3× Gel buffer

3 ml

10 ml

Glycerol−2.4 ml

dH2O

8 ml

7.6 ml

10 % APS

90 μl

100 μl

TEMED

9 μl

10 μl

1-3) Sample preparation
Prepare the protein sample according to the following table.
Protein sample

15 μl

4× Sample buffer

5 μl
20 μl

Heat at 95°C for 5 min, and then put the tube on ice.
1-4) Electrophoresis of the protein samples and DynaMarker® Protein MultiColor Stable, Low Range.
Set the Polymerized acrylamide gel in the electrophoresis apparatus, then pour the 1× Anode buffer and1× Cathode buffer. Load the protein sample and 5 μl of DynaMarker® MultiColor Stable, Low Range into awell and run the gel at 100−200 V.
2) Transfer onto membrane2-1) Preparation of reagents・Transfer buffer (Towbin buffer)
25 mM Tris, 192 mM Glycine, 20 % MeOH
・Nitrocellulose membrane (0.2 μm pore size).
Note: Transfer efficiency differs depending on the type of membranes and buffers, because the physical properties of small
molecule proteins greatly vary depending on kinds of proteins.

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BioDynamics Laboratory Inc.
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Ver.1.1

2-2) Semi-dry transfer−−

Paper
Gel
Membrane
Paper++
Figure 1, Semi-dry transfer
2-2-1) Prepare six sheets of blotting paper and a sheet of nitrocellulose membrane.
2-2-2) Soak the blotting paper and nitrocellulose membrane with transfer buffer for 10 min.
2-2-3) Place the three sheets of blotting paper on the anode platform of the transfer cell.
2-2-4) Place the membrane on top of the blotting paper.
2-2-5) Transfer the gel from the glass plate to the top of the membrane and press out any air bubbles.
(*It is important to make sure that there are no air bubbles between the membrane and the gel.)
2-2-6) Place the other three sheets of blotting paper onto the gel and set the cathode assembly.
2-2-7) Transfer for 60 min at 2 mA/cm2.
2-2-8) After ensuring the marker has transferred successfully onto the membrane, remove the membranefrom apparatus.
2-2-9) Rinse the membrane in PBS buffer.
3) Detection3-1) Boil the membrane for a few minutes (3～5 min.) in PBS buffer (3)3-2) Block with TBS based protein free blocking agent (e.g. Pierce® Protein-Free T20 (TBS) BlockingBuffer*) for 1 hr
3-3) Wash 3 times for 5 min in TBS-T(0.05% Tween20)3-4) Incubate with anti β-Amyloid antibody (mouse monoclonal) overnight at 4 °C
3-5) Wash 3 times for 5 min in TBS-T(0.05% Tween20)3-6) Incubate with biotinylated anti mouse IgG antibody for 1 hr at room temperature.
3-7) Wash 3 times for 5 min in TBS-T(0.05% Tween20)3-8) Incubate with VECTASTAIN Elite ABC Standard kit**(Please refer to the manual.)
3-9) Wash 3 times for 5 min in TBS-T(0.05% Tween20)3-10) Stain with DAB Peroxidase substrate kit***.
(Please refer to the manual.)*: Pierce® Protein-Free T20 (TBS) Blocking Buffer is a product of Thermo Fisher scientific, Inc.
**: VECTASTAIN Elite ABC Standard kit is a product of Vector laboratories, Inc.

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***: DAB peroxidase substrate kit is a product of Vector laboratories, Inc.

8.7 kDa
3.9 kDa
1.7 kDa← β-Amyloid (1-42)
4.5 kDa

Figure 2, Detection of β-Amyloid (1-42)

Reference(1) Hermann schägger and Gebhard Von Jagow. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Anal. Biochem. 166,
368-379 (1987).
(2) Hermann schägger. Tricine-SDS-PAGE. Nature Protocols. 1, 16-22 (2006)(3) Nobuo Ida, et.al. Analysis of Heterogeneous βA4 Peptide in Human Cerebrospinal Fluid and Blood bya Newly Developed Sensitive Western Blot Assay. J. Biol. Chem. 271, 22908-22914 (1996)

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Ver.1.1