www.toyobo.co.jp/e/bio

F1029K

Instruction manual PVDF Blocking Reagent for Can Get SignalTM 2004

PVDF Blocking Reagent

for Can Get SignalTM
NYPBR01 500 mL
Store at 4 °C.

Contents[1] Introduction[2] Components[3] Protocol[4] Application data
[5] Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precautions and follow safety guidelines while using this kit.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/biotech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description

PVDF Blocking Reagent for Can Get SignalTM is a high performance blocking reagent
optimized for Western blot. The reagent consists of a synthesized polymer, with no protein
components. The reagent can be used efficiently with Can Get SignalTM Immunoreaction
Enhancer Solution.

Features– PVDF Blocking Reagent for Can Get SignalTM has been optimized for use together withCan Get SignalTM Immunoreaction Enhancer Solution (Code No. NKB-101) for Western
blot analysis.
– The reagent is suitable for detection of phosphorylated proteins, because it does notcontain any protein components.
– The reagent minimizes the masking effects of low signal intensities, whereas
conventional blocking reagents (e.g., non-fat milk and gelatin) can mask Western blot
protein signals.

[ 2 ] Components

This reagent should be stored at 4°C.
PVDF Blocking Reagent for Can Get SignalTM

500 mLNotes:- The reagent contains 0.1% sodium azide.

[3]

ProtocolPDVF Blocking Reagent should be used directly to block non-specific protein binding on
Western blots.
The standard protocol is as follows:(1) Wash transferred membranes in Wash Buffer (e.g., TBS-T: TBS/0.1% Tween 20) for
5 min. while shaking.
(2) Replace Wash Buffer with PVDF Blocking Reagent for Can Get SignalTM.
(3) Incubate at RT-37°C for 1 hr, or at 4°C overnight.
(4) Rinse the membrane in Wash Buffer.
(5) Wash the membrane in Wash Buffer for 15 min, and twice for 5 min. per wash whileshaking.
(6) Proceed to the primary antibody reaction step.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio

Notes:- PVDF Blocking Reagent for Can Get SignalTM can be applied to PVDF (polyvinylidene
difluoride) and nitrocellulose membranes used for Western blot analysis. The reagent
works more efficiently on PVDF membranes.
- This reagent has been optimized for Western blot analysis with Can Get SignalTM
Immunoreaction Enhancer Solution. Although this reagent can be used for conventional
Western blot analysis, blocking efficiency may be decreased.
- Because this reagent contains 0.1% sodium azide, residual sodium azide may inhibit HRP
activity. Therefore, the washing step should not be skipped after the blocking step.

[ 4 ] Application data

Example 1<Assay conditions>
SDS-PAGE: 8-16% polyacrylamide gel, 15 mA × 90 min.
Transfer: 0.8 mA/cm2 at RT for 60 min. (semi-dry method)
Blocking: RT for 60 min.
Sample: HeLa cell lysates 2 × 104 cells/well (1/1), 4n dilution (1/4, 1/16)
Primary antibody: rabbit anti-ERK2 (C-14) antibody (0.1 ng/μL) in Can Get SignalTM
Solution 1
Secondary antibody: HRP-conjugated anti-rabbit IgG antibody (0.02 ng/μL) in Can Get
SignalTM Solution 2Detection reagent: ECL Plus (GE Healthcare)<Result>

PVDF Blocking Reagent for Can Get SignalTM successfully increased protein signal
intensity and reduced non-specific background staining.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

2

www.toyobo.co.jp/e/bio<Assay conditions>
SDS-PAGE: 8-16% polyacrylamide gel, 15 mA × 90 min.
Transfer: 0.8 mA/cm2 at RT for 60 min. (semi-dry method)
Blocking: RT for 60 min.
Sample: HeLa cell lysates 2 × 104 cells/well (1/1), 4n dilution (1/4, 1/16)
Cells were stimulated with EGF.
Primary antibody: mouse anti-p-ERK2 (E-14) monoclonal antibody (0.2 ng/μL) in Can Get
SignalTM Solution 1
Secondary antibody: HRP-conjugated anti-mouse IgG antibody (0.01 ng/μL) in Can Get
SignalTM Solution 2
Detection reagent: ECL Plus (GE Healthcare)<Result>
Simulation by
EGF (500 ng/ml, 5 min)
(-)

(+)

1/1 1/4 1/16

1/1

1/4 1/16

The distinct bands (p-ERK1: 44 kDa, p-ERK2: 42 kDa) were successfully detected withminimal background staining.

[ 5 ] Related productsProduct name

Can Get Signal TM
Solution 1 for primary antibody
Solution 2 for secondary antibody
Can Get Signal TM
Solution 1 for primary antibody
Can Get Signal TM
Solution 2 for secondary antibody

Package

Code No.

250 mL each
50 mL each

NKB-101
NKB-101T

250 mL

NKB-201

250 mL

NKB-301<Manufacturer>

Example 2
JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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