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TMB Enhanced One Component HRP Membrane
Substrate
Catalog Number T9455
Storage Temperature 2-8 °C
TECHNICAL BULLETIN
Product Description
TMB Enhanced One Component HRP Membrane
Substrate is a ready to use solution containing
3,3’,5,5’-tetramethylbenzidine in a mildly acidic buffer.
The substrate reacts with peroxidase to form an
insoluble permanent dark blue/purple color on the
membraneor surface to which it is applied. This
enhanced formulation extends the dynamic range of
detection, especially athigher concentrations of
peroxidase where other substrates may exhibit loss of
signal due to washout effects. This substrate is not
recommended for microwell or immunohistochemical
applications. Unreacted substrate should be faint
yellow to pink in appearance.
Available Package Sizes
Catalog Number
T9455-100ML
T9455-1L
Precautions and Disclaimer
This product is for R&D use only, not for drug,
household, or other uses. Please consult the Material
Safety Data Sheet for information regarding hazards
and safe handling practices.
Storage/Stability
TMB Enhanced One Component HRP Membrane
Substrate is stable for 18 months from date of
manufacture when stored at 2-25°C. Storage is
recommended at 2-8 °C. This product is stable for 4
years at 2-8° C. Thissubstrate is light sensitive and
should be protected from direct sunlight and UV
sources. Over time, some flocculent material may
appear; however, this does not affect product
performance.
Procedure
TMB Enhanced One Component HRP Membrane
Substrate is supplied as a ready to use solution. The
product should be allowed to equilibrate to room
temperature (25°C) prior to use. For use, completely
cover the membrane surface with substrate. The
substrate system will react with sites on the membrane
containingperoxidase, producing an insoluble
permanent dark blue reaction product. To stop the
reaction, rinse the membrane with reagent quality
water. The reaction should be monitored and read
before the background color becomes too intense
which may result in diminished contrast between the
positive and background staining. Dilution of the
substrate is not recommended. To reduce the intensity
of a reaction, it is recommended that the antibodies or
conjugates be diluted.
ImmunoBlot Application:
brate TMB Enhanced One Component HRP
Membrane Substrate(T9455) to room temperature
if stored at 2-8 °C.
er proteins to membrane as per your
established protocol.
membrane from the transfer apparatus
and place in blocking buffer for 30–60 minutes at
room temperature or overnight at 4° C.
blocking solution, add primary antibody to
the membrane, and incubate for 30–60 minutes at
room temperatureor overnight at 4° C.
the primary antibody is directly conjugated to
HRP, proceed to step 7.
using secondary antibody, wash the membrane
3X with either TBS or PBS containing 0.05%
®
TWEEN-20. Remove the wash solution and
incubate with secondary antibody for 30–60
minutes at room temperatureor overnight at 4° C.
the membrane 3X with either TBS or PBS
containing 0.05% TWEEN-20.
all solution and carefully blot off excess
with paper towel.
enough room temperature TMB Enhanced
One Component HRP Membrane Substrateto
cover the entire membrane; monitor the reaction
time carefully. Full color should develop within 10-
30 minutes, but timing should be optimized for each
assay. To stop the reaction, wash the membrane at
least 2X in distilled water.
RG,IDC,PHC 07/12-1
©2012 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other
countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their
particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at
/or on the reverse side of the invoice or packing slip.
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