Technical Data Sheet
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TEL : 886-2-2723-0886 www.recenttec.com
DIGE kit
Catalog：R13-DG03-1P
Package：
Components
Volume
Package
Cy2
400 pmole
1 bottle
Cy3
400 pmole
1 bottle
Cy5
400 pmole
1 bottle
DMF
20µl
1 bottle
Storage：This solution can be stored at 4°C for 1 year.
Description
Sensido DIGE kit is one of 2-D Fluorescence Difference Gel
Electrophoresis (2-D DIGE) specific CyDye probes. The DIGE kit
includes three different CyDye used to label proteins that are
subsequently separated using 2-Dimensional gel electrophoresis.
Each of the individual samples can then be visualized independently
by selecting the individual excitation and emission wavelengths for
each CyDye when fluorescence scanning. Sensido DIGE kit is ready
to use in protein minimal labeling and cheaper than other brands.
Advantages
1. Ready to use
Add 50 μg protein sample in each CyDye for minimal labeling
2. Reduce the cost of purchasing DIGE material
Cost down the experimental CyDye material of 2-D DIGE
Safety
Information
Please wear gloves, lab coat and gaggles while operating. Prevent
contact product directly. In case of contacting, wash with largeamount
of water.
Materials Needed
but Not Provided
• Sample wash buffer: 10 mM Tris (pH 8.0), 5 mM Magnesium Acetate
• Lysis buffer: 30 mM Tris, 7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS.
Adjust to pH 8.5 with diluted HCl
• Lysine: 10 mM L-Lysine (Sigma™ L5626)
• pH indicator strips: Sigma pH test strips pH 4.5–10.0 (Sigma P4536)
• 2X Sample buffer: 8 M Urea, 130 mM DTT, 4% (w/v) CHAPS, 2%
(v/v) IPG buffer
• Rehydration buffer: 8 M Urea, 4% (w/v) CHAPS, 1% (v/v) IPG buffer,
3 mM DTT, and a few grains Bromophenol Blue
For research use only. Not for use in diagnostic procedures.
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Technical Data Sheet
Instruction
A. Sample preparation
1.Collect the appropriate amount of sample (5~10×107 cell line) by
centrifugation at 500xg, 4°C for 5 minutes. Discard the supernatant
carefully.
2.Wash the sample pellet with 5 mL of sample wash buffer and repeat
3 times.
3.According to sample volume, add into 2-3 folds volume of lysis buffer.
4.Extract protein by freeze and thaw (repeat 3 times), then centrifuge
at 12,000xg, 4°C for 10 minutes and transfer supernatant to a new
tube on ice.
5.The recommended concentration of the protein lysate is between 5
and 10 mg/mL. Samples containing from 1 mg/mL to 20 mg/mL have
been successfully labelled using the protocol below.
Note: Check that the pH of the cell lysate is still at pH 8.5 by spotting
1 μL cell lysate on a pH indicator strip. If the pH of the cell lysate
has fallen below pH 8.0 then the pH of the lysate will need to be
adjusted before labelling.
B. Protein minimal labeli
1.Add 1 μL DMF in amber screw tubes of DIGE kit.
2.Vortex well and spin down. Repeat this step three times.
3.Add a volume of protein sample equivalent to 50 μg to the amber
screw tube.
4.Mix well and spin briefly. Then leave on ice for 30 minutes in the
dark for labeling reaction.
5.Add 1 μL of 10 mM lysine to stop the reaction. Mix by pipetting and
spin briefly.
6.Leave for 10 minutes on ice in the dark.
7.Samples can be stored for at least three months at -70°C in the dark.
C. Loading samples onto IPG strips
1.Add an equal volume of 2X sample buffer into the DIGE kit labeled
protein samples and leave on ice for 10 minutes.
2.Pool all the DIGE kit labeled protein samples.
3.According to IEF instrument suggestion, add enough volume of
rehydration buffer to execute IEF.
For research use only. Not for use in diagnostic procedures.
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Technical Data Sheet
Troubleshooting
Problem
Possible cause
Remedy
The pH of the protein 1. The lysis of the
1. Increase the
lysate is less than
cells has caused a
buffering capacity
pH 8 before labeling.
drop in the pH.
of the Lysis buffer
2. The sample wash
to 40 mM Tris (50
buffer was not
mM is the recommcompletely removed
ended maximum
for Tris)
2. Increase the pH of
the lysis buffer by
the addition of a
small volume of 50
mM NaOH. Or add
an equal volume of
the lysis buffer that
is at pH 9.5.
For research use only. Not for use in diagnostic procedures.
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