Desired Antibody Diluent
to Enhance Immunoreaction
at Western Blotting, Dot Blotting and ELISA
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Wako Cat. No. 294-68601 (for 2 assays)
290-68603 (for 10 assays)
298-68604 (for 40 assays)
Wako has launched an accelerator to optimize antigen-
antibody reactions for Western blotting, dot blotting,
enzyme-linked immunosorbent assay (ELISA), etc.
Immuno-enhancer aids in obtaining high signal-to-noise
(S/N) ratio.
This kit consists of ready-to-use solutions such as
Reagent A as the diluent of the primary antibody, and
Reagent B as the diluent of the secondary antibody diluent.
Features
Enhances Immunoassay Signals, Effectively
High S/N Ratio
Applicable to Ready-to-Use Antibody Diluent
Application-1
A549 cell Lysate 5 µg (1) or 10 µg (2) were
subjected to SDS-PAGE and blotted onto
nitrocellulose membranes.
They were blocked and Western blot.
3% skim milk in TBS-T solution was used as the
control.
Antibodies:
Primary Antibody: Anti EB1, Rabbit (1:500),
2 hour-reaction
Secondary Antibody: HRP conjugated Anti
rabbit IgG (1:7000), 1 hour-reaction
Exposure time:
10 seconds
Procedures
Western blotting
ELISA, using labeled antibody
SDS-PAGE
Transfer to a membrane
Blocking
Reaction with Primary Antibody,
diluted with Immuno-enhancer Reagent A
Reaction with Secondary Antibody,
diluted with Immuno-enhancer Reagent B
Detection
Coat a microplate with antibody
Blocking
Antigen (Samples),
diluted with Immuno-enhancer Reagent A
Reaction with labeled Antibody
(=Secondary Antibody),
diluted with Immuno-enhancer Reagent B
Detection
Application-2
Western blotting
HeLa Cell Lysate, diluted by 1/1, 1/2, 1/4 and 1/8 were subjected to
SDS-PAGE and blotted onto a membrane, blocked, followed by Western
blotting.
TBS-T solution was used as the control.
Antibodies:
Primary antibody: Anti Actin, Goat (0.5 g/mL), 1 hour-reaction
Secondary antibody: Anti goat-IgG-HRP antibody (1:10,000 dilution)
1 hour-reaction
Exposure time:
5 minutes
Application-3
Western blotting
~ Achieved LC3-I detection in Atg7+ ~
Cell extract was separated by 12.5% SDS-PAGE, transferred onto PVDF
membrane, followed by Western blotting the primary antibody diluted with
Immuno-enhancer Reagent A. 20 mM Tris-HCl (pH 7.5)-0.15 M NaCl-0.1%
NaN3 containing 1% BSA was used as the control. The secondary antibody
was diluted with 20 mM Tris-HCl (pH 7.5)-0.15 M NaCl-0.05% Tween 20.
Antibodies:
Primary Antibody: Anti Rat LC3 antiserum, rabbit
Secondary Antibody: Anti rabbit-IgG-HRP antibody
Exposure time:
15 seconds
Generally, LC3-I form is shown in Atg7-de cieny cell and LC3-II is mainly
detectable in Atg7+. To detect LC3-I in Atg7+, it is necessary to lengthen
the exposure time. By using Immuno-enhancer, achieve LC3-I detection,
even if the shorter exposure time.
(Data was provided by Dr. Takashi Ueno, Dep. of Biochemistry, Juntendo Univ. School of Medicine.)
Description
Wako Cat. #
Pkg. Size *
Kit Contents
Reagent A,
for Primary Antibody
Reagent B,
for 2nd Antibody
Immuno-enhancer
<for blotting>
294-68601
290-68603
298-68604
1 bottle 10 mL
1 bottle 50 mL
1 bottle 200 mL
1 bottle 10 mL
1 bottle 50 mL
1 bottle 200 mL
for 2 assays
for 10 assays
for 40 assays
* : Each package size shows an assay number when 5 mL each is used per assay.
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