Good transfer of protein standard bands onto the nitrocellulose membrane.
Protein Standards were separated on a 4-20% GenScript Express
TM
Plus
PAGE Gel (Bis-Tris). After electrophoresis, the gel was blotted using the
e
Blot
TM
protein transfer system for 11 min as described in this manual.
Lane 1 and 2, Invitrogen Pre-Stain protein marker (5
μ
L, MW: 4-250 kDa),
Lane 3 and 4, Bio-rad Pre-Stain protein marker (10
μ
L, MW: 10-250 kDa),
and Lane 5-9, NEB All Blue Pre-Stain protein marker (0.3, 0.6, 1.3, 2.5, and
5
μ
L, MW: 10-230 kDa).
- 22 -
e
Blot
TM
Protein Transfer System
www.genscript.com
- 21 -
e
Blot
TM
Protein Transfer System
www.genscript.com
Examples of Results
Troubleshooting
Problem
The right Status Light doesn’t
flash during blotting process.
The left and right Status
Lights flash simultaneously.
Inefficient transfer
Empty spots on the membrane
Cause
Incomplete electric circuit due
to improper assembly of the
transfer pads.
Excessive current is flowing
through the Device.
1. Salt built-up on plate electrodes
2. Membrane insufficiently
equilibrated in
e
Blot
TM
Equilibration
buffer
3. Incorrect transfer conditions or
insufficient transfer time
4. PVDF membrane was not prewet
with methanol
5. Confusion of the
e
Blot
TM
Anode
Pad and Cathode Pad
Air bubbles trapped between gel
and membrane prevent the
transfer of proteins.
Solution
Ensure the transfer stack is
assembled correctly: use the
e
Blot
TM
Anode Pad first followed
by the membrane, the pre-run gel,
Gel Window and
e
Blot
TM
Cathode
Pad.
Check the transfer stack and
ensure Gel Window covered
correctly on the gel.
1. Clean the titanium cathode
plate, graphite anode plate
with a wet cloth or paper
tissue followed by a dry one
to remove any insoluble
salts.
2. Equilibrate membrane in
e
Blot
TM
Equilibration
buffer before transfer.
3. Use a gel of lower
concentration to separate
high molecular weight proteins.
Increase the transfer time in
5-second increments.
4. Pre-wet PVDF membrane
with methonal before
transfer.
5. Ensure the transfer stack is
assembled correctly:
Bottom-e
Blot
TM
Anode Pad
(yellow), Top-
e
Blot
TM
Cathode
Pad (white).
When assembling transfer stack,
use the small shovel supplied with
the device to remove any air
bubbles between the gel and the
membrane.
An Express™ PAGE Gels 8-16% (GenScript MG816W12) was blotted using
the
e
Blot
TM
. 10.0, 5.0, 2.5, 1.25 0.62, 0.31 and 0.16
μ
g of HeLa cell lysate
were loaded respectively. Proteins on the Nitrocellulose membrane were
detected using THE
TM
alpha Tubulin Antibody (GenScript, A01410), THE
TM
beta Actin Antibody (GenScript, A00702) and GAPDH Antibody (GenScript,
A01632). The secondary antibody was Anti-MOUSE IgG (H & L) (GOAT)
Antibody Peroxidase Conjugated (Rockland, 610-1302). The signal was
developed with LumiSensor
TM
HRP Substrate Kit (GenScript, L00221V500)
Westen blot result after
using the eBlot
TM
for
protein transfer
Result Using Nitrocellulose
1
2
3
4
5
6
7
8
9
kDa)
kDa)
kDa)
