The reporter molecule will allow the visualization of the target protein at the end of the assay. Primary antibodies conjugated to a reporter do not require secondary antibody usage. Wash steps with a mild detergent containing buffer are typically performed after antibody incubations to remove any non-specific binding.
Western blot experiments (much like ELISA immunoassays) can
be performed in several formats, most requiring a conjugated secondary antibody
to act as the reporter molecule. Reporter molecules include horseradish peroxidase
and alkaline phosphatase
enzymes as well as fluorophores.
reporter enzymes are used, chromogenic or luminescent substrates can be applied for detection. Chromogenicsubstrates are used in colorimetric assays since they result in a measurable
color change in the presence of an enzyme-antibody complex bound to specific
analytes. For WB using horseradish peroxidase (HRP) in colorimetric detection, TMB and DAB substrates
are commonly used. Alkaline phosphatase (AP) chromogenic substrates include BCIP/NBT, which usually
exhibit the highest sensitivity and reliable detection of AP activity.
Chemiluminescent substrates in the other hand offer several
advantages over the chromogenic
substrates. Mainly, these systems
are significantly more sensitive for detection of enzymatic activity without
the use of radioactive isotopes, luminescent detection typically happens within
few minutes and the signal is more amenable to quantification because it
requires the use of digital charge-coupled devices (CCD) for detection that
allow for a wide dynamic range using prolonged exposure.
Fluorophore reporter molecules do not require substrate, but they do
require specialized equipment for data collection. Fluorescent detection is
suitable for multiplex WB
experiments where multiple targets can be detected in the same assay using fluorophore
conjugates with non-overlapping emission spectra. Fluorescent WB is also ideal for quantitative
analysis since detection allows for wide dynamic ranges and signal
The choice of WB membrane
depends on the type of experiment to be performed. Most commonly used are nitrocellulose or
polyvinyldifluoride (PVDF). Nitrocellulose is easy to use and provides suitable
data for most common enzymatic reporter experiments. Low fluorescent PVDF membranes
are recommended for fluorescent Western blot applications.
Please find detailed Western blotting Protocols and Resources here.
As application-specific guidelines and standards for validating research antibodies increasingly becomes a subject of scrutiny
by the scientific community, multiple approaches can (and should) be
implemented to demonstrate the specificity of a primary antibody with adequate
robustness. One of such approaches is the confirmation of target specific
antibody properties by the inclusion of multi-lysate panels from cells known to
express or not the target of interest based on genomic and proteomic studies.
The appropriate negative controls for cells naturally producing the target are
the same cells in which the abundance of the target is selectively altered by
chemically stimulation or genetic approaches.
Rockland Immunochemicals routinely scrutinize the
specificity or its primary antibodies by assessing their performance in
multi-lysate Western blots. A variety of conditions are evaluated for each
target so that specificity, sensitivity and reproducibility can be determined.
This ensures reliable performance and confirm lot-to-lot consistency.
To learn more about experimental details
regarding multi-lysate Western blotting please visit Rockland's Protocols Page.
Immunoprecipitation (IP) is one of the most widely
used approaches for antigen purification and detection. The approach uses
antigen-specific antibodies to isolate from a complex protein mixture an
antigen of interest that is subsequently analyzed by Western blotting in order
to assess the relative amount and size of the target antigen itself and/or
Many of Rockland's antibodies have been validated for use in In-Cell Western (ICW) assays. Since ICW assays are related to immunofluorescence most antibodies that have been validated and approved for these aforementioned immunoassays can be similarly used with ICW assays. Antibodies suitable for cell based immunoassays can be identified under the applications field of the Rockland website catalog. Other reagents generally required in ICW include:
When performing a Western blot, the blocking buffer should not be
blocking buffer fills in the locations on the membrane that can still bind
protein and cause background if not treated. The critical reagent in a blocking
buffer is protein, where the protein is non-antibody reactive. Popular blocking
proteins include non-fat dried milk (NFDM), BSA, casein, and combinations
thereof. Of note, a single blocking agent may not be sufficient for all western
applications. Some blocking agents can interfere with primary antibody
activity, or may not be compatible with the reporter system in use, or produce
undesired auto-fluorescence. Rockland develops several blocking buffer reagents
suitable for all Western blot applications, including BLOTTO-NFDM and BSA for
standard applications, and a specially formulated blocking buffer forfluorescent Western blotting.
Secondary Antibody Conjugates
Secondary antibody conjugates are ideal for Western
blotting. When choosing a secondary antibody conjugate for an antibody
assay consideration must be given to target species, conjugate (i.e. peroxidase,
FITC, Biotin) and host species. In addition to standard secondary antibodies,
Rockland offers pre-adsorbed secondary antibodies which are suitable for
detection methods where cross-reactivity may be an issue.
Peroxidase Conjugated Secondary Antibodies
Reporter enzymes are used extensively in molecular biology because they allow visualization or detection of immune complexes. Horseradish Peroxidase (HRP) is a widely used reporter enzyme, and depending on the substrate it can yield a chromogenic, or luminescent product (chemiluminescence). Alkaline phosphatase is also used, most typically as the reporter in chromogenic Western blot assay format.
Rabbit IgG (H&L) Antibody Peroxidase Conjugated Pre-Absorbed
Alkaline Phosphatase Conjugated Secondary Antibodies
Antibodies Conjugated to Alkaline Phosphatase
(AP or Alk Phos) are used in the detection of proteins in Western blotting and ELISA
immunoassay procedures. The alkaline phosphatase (AP) catalyzes colorimetric
reactions using BCIP/NBT Substrates or FemtoMax chemiluminescent substrate.
Secondary Antibody conjugates are conjugated to the highest grade of alkaline
phosphatase using Rockland’s proprietary technology.
Fluorescent Secondary Antibody Conjugates
Trueblot® IP/ Western blot
IP Western blots provide highly specific results,
yet often suffer from heavy/light chain blotting, contamination, and ongoing
products solve nearly all of
these problems through increased sensitivity, less background noise, and
Rockland produces several luminol based
substrates with chemiluminescence for the detection of horseradish peroxidase
(HRP). PicoMax™ and FemtoMax™ are designed for high
performance in Western blotting and are functional on both nitrocellulose and
PVDF membranes. FemtoMax™ produces chemiluminescence and allows for the
detection of down to femtogram (10-15) amounts of antigen. Detection
methods may include photographic film or other imaging methods, including
highly sensitive CCD camera based systems.
blotting substrates are available from Rockland in a variety of specifications
and formats. The appropriate substrate choice depends on the enzyme label,
desired sensitivity and form of signal or method of detection needed.
Chromogenic Peroxidase Substrates:
The Peroxidase reaction with our TMBM
substrate produces a water-soluble blue product that can be precipitated onto a
membrane. The precipitating product produces blue to dark blue bands
in the enzyme location. TMBM is well suited to applications that require
high signal-to-noise. DAB is another peroxidase substrate and yields a
brown precipitate in the presence of HRP and peroxide.
Chromogenic Alkaline Phosphatase
The NBT/BCIP reagent is also commonly
used in chromogenic Western blot immunoassays. NBT serves as an oxidant
and BCIP as the alkaline phosphatase substrate. Together NBT and BCIP
form reactants in the presence of alkaline phosphatase which yields a dark
purple to black, water-insoluble, precipitant product providing strong
all our substrates
Western blot kits may be available for your assay, simplifying your reagent needs. Rockland offers Kits for chemiluminescence, fluorescent, and chromogenic immunoassay formats. Our Western blotting kits are configured with simple and easy to use protocols for both beginner and expert users alike. Kits are species specific for detection of mouse or rabbit primary antibodies, and come ready as a format specific package that includes membrane blocking reagent, washing buffers, secondary antibodies and substrate (if required). Some of our more popular kits include FentoMax kits for chemiluminescent applications, infrared (IR) and Dylight™ kits for detection on fluorescent Western blot protocol imaging systems.
Browse all of our Western blot Kits
Loading controls are important for correct interpretations of Western blots. Control Antibodies are used to normalize the levels of protein detected by confirming that protein loading is the same across the gel. The expression levels of the loading control should not vary between the different sample lanes.
Rockland has developed Revitablot™ Western Blot Stripping Buffer, which contains solutions in a proprietary combination to enhance the removal of bound antibodies from western blot membranes for repeated use. The proprietary formulation of the solution ensures high stripping efficiency with low backgrounds.
Revitablot™ Western Blot Stripping Buffer (500mL) is a uniquely formulated, ready-to-use reagent specifically designed for western blotting. Revitablot™ is a faster and more efficient at stripping primary and secondary antibodies, allowing blots to be stripped multiple times for the repeated use of membranes. This solution can be used at room temperature and only requires 5-20 minutes of incubation time to strip the membrane. Revitablot™ Western Blot Stripping Buffer (50mL) is gentle and effective on both nitrocellulose and PVDF western blot membranes.
Rockland Immunochemicals Inc.Limerick, PA 19468E-mail: firstname.lastname@example.orgPhone: 800.656.7625