Introduction:
Western
Blot is widely used to detect and compare proteins from a complex
mixture utilizing antibody detection on a membrane. Chemiluminescence
has become an easy and sensitive method of detection compared to other
analysis. Because of the nature of chemiluminescence detection, it is
possible to reprobe the separated protein mixture on the membrane.
Conventionally, Western blots have been stripped using extremely harsh
conditions that may alter the antigen for subsequent immunoprobing. Gene
Bio-Application, Stripping buffer is a novel formula provides a gentle
method of removing primary and secondary antibodies membranes that
allows several reprobings on the same membrane.
 
PRODUCT CATALOG REFERENCE
|
Cat.#
|
Description
|
|
ST010
|
SDS removing buffer, 500 ml
|
|
ST013
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SDS removing buffer, 60 ml
|
Protocol:
IMPORTANT:
Optimization of incubation time is essential for optimum results.
IMPORTANT:
If the blot cannot be stripped immediately after chemiluminescence
detection, the blot can be stored in PBS or TBST at +4oC until
the stripping procedure is to be preformed.
-
Place the blot to
be stripped in the Stripping Buffer. Add Stripping Buffer, to
fully immerse the blot.
-
Incubate the blot
in the Striping buffer for 5-15 min at room temperature with
strong shaking.
-
IMPORTANT:
In general, higher affinity antibodies or large quantities of
detected protein will require longer incubation time for
stripping.
-
Empty the
Stripping Buffer.
-
To wash, add 0.3
ml of dH2O and shake vigorously.
-
Repeat Steps 4
five more times.
Complete
removal of the HRP label monitoring: After Step 6
incubate the membrane with fresh chemiluminescence reagents and
expose to film. If no signal is detected with a 5 min exposure,
the HRP conjugate has been successfully removed from the antigen
or primary antibody.
Complete removal of
primary label antibody monitoring: After Step 6
incubate the membrane with the HRP-labeled secondary antibody,
followed by a wash in wash buffer. Incubate the membrane with
fresh chemiluminescence reagents and expose to film. If no
signal is detected with a 5 min exposure, the primary antibody
has been successfully removed from the antigen.
IMPORTANT: Analysis
of the successful removing of immunoprobes is recommended to
prevent removal of the antigen or the unsuccessful removal of
the antibodies.
If signal is
detected in the two experiments describe above, place the blot
back into Stripping Buffer for additional 5-15 min.
-
After it has
been determined that the membrane is free of the immunodetection
reagents, a second immunoprobing can take place. Start the
second immunoprobing with reblocking of the blot.
IMPORTANT:
The blot can be stripped up to 5 times. However, longer exposure
times or more sensitive chemiluminescence substrate. Actually,
reprobings may result in a decrease in signal if antigen is
labile. Analysis of the individual system is required.
Representative Result
Stripping
immonoblot with Stripping Buffer and the reprobing the immonoblot
Stripping of Westren blot by Stripper Buffer and ReWestren blot:
(A) Whole HeLa extract was loaded on 10% SDS-PAGE as
the indicated amount. After gel separation it was blotted on a
nitrocellulose membrane by a semi dry blotter. The membrane was blocked
by 1% fat-milk for 1 hour, washed for 15 min and two more times for 5
min with TBST. The first immunoblot was preformed with rabbit anti actin
antibody for 1 hour (dilution 1: 500) and washed. Goat anti rabbit IgG
conjugated to HRP was used as the secondary antibody (dilution 1: 5000)
for the chemiluminescence detection. The immunoblot was exposed to film
for 15 min. The scanned aoutoradiogram is presented. (B)
The first immunoblot was stripped by immerse the blot in the Stripper
Buffer for 7 min, and washed for 5 min, 5 times. After stripping, the
immunoblot was immersed in chemiluminescence reagent and the blot was
exposed to film for 60 min. The scanned aoutoradiogram is presented.
(C) Reprobing the first immunoblot (second immunoblot)
was blocked again by 1% fat-milk for 1 hour, washed for 15 min and two
more times for 5 min with TBST. The second immunoblot was preformed with
rabbit anti eIF2 antibody for 1 hour (dilution 1: 500) and washed. Goat
anti rabbit IgG conjugated to HRP was used as the secondary antibody
(dilution 1: 5000) for the chemiluminescence detection. The immunoblot
was exposed to film for 15 min. The scanned aoutoradiogram is presented.
(*) Marks are unspecific detections.
NOTES:
1. All
incubation was made at room temperature
2. All
incubation was made in shaker. |
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+32 (0) 16
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