Western Blotting

Western blot (WB) or immunoblot is a workhorse immunoassay for most labs, used to demonstrate antibody specificity, confirm gene expression, detect post-translational modifications and diagnose diseases among others. Specific detection of bands corresponding to the protein of interest result from successively probing your blot with Rockland’s primary and conjugated secondary antibodies. Reagents for Western blotting are available from Rockland as standalone products, but also as easy to use preassembled Western Blotting kits.



Western blot is a widely used method for detection of a specific protein in a complex matrix such as cell or tissue lysate (i.e. protein extracts).  Western blot protocol uses gel electrophoresis (SDS-PAGE or native PAGE) to separate proteins according to molecular weight. The proteins are then transferred from the gel onto a membrane (typically nitrocellulose or PVDF).  The membrane is blocked with a protein blocking buffer to prevent nonspecific binding and then probed using a primary antibody to detect the protein of interest followed by an incubation with a secondary antibody conjugated to a reporter molecule. 


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.

western blotting procedure  

When 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 normalization.


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.


Multi-lysate Western blotting


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.  



Histone H3 Antibody
EGFR Antibody



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.

Western blotting for Immunoprecipitation (IP)


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 target-associated proteins.  



WB-3   Analysis of immunoprecipitated proteins by immunoblotting can be complicated because the re- agent used to detect the WB staining antibody will often bind to the heavy and light chains of the precipitating antibodies. As described below, this problem can be easily corrected by using Rockland’s TrueBlot® products that selectively bind staining antibodies only through increased sensitivity, less background noise, and enhanced accuracy.  



In-Cell Western


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: 




Alpha-Tubulin Antibody 


Blocking Buffers for Western blotting


When performing a Western blot, the blocking buffer should not be overlooked. The 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 Antibodies for 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.


 WB-5       WB6

RABBIT IgG (H&L) Antibody Alkaline Phosphatase Conjugated



Fluorescent Secondary Antibody Conjugates

Rabbit IgG (H&L) Antibody DyLight™ 

 Mouse IgG (H&L) Antibody DyLight™


Rockland conjugates a broad group of secondary antibodies to many of the classic and next generation of fluorescent markers including fluorescein, Texas Red, Phycoerythrin. Rockland also produces many next generation flurochrome dyes. These are designed for detection of primary antibodies in multiplex, multi-color analysis.  Next generation fluorochrome conjugates (Atto-tec dyes, DyLight dyes) offer superior absorption (high extinction coefficient), high fluorescence quantum yield and superior high photostability. All of the conjugates are ideal for various immunofluorescence based assays including fluorescent Western blotting, immunofluorescence microscopy, FLISA, and more. 

Browse all of our secondary reporter antibodies

Trueblot® IP/ Western blot

IP Western blots provide highly specific results, yet often suffer from heavy/light chain blotting, contamination, and ongoing interference. TrueBlot® products solve nearly all of these problems through increased sensitivity, less background noise, and enhanced accuracy.

TrueBlot® reagents enable you to generate clear, best-quality data in your Immunoprecipitation and Western blot protocols. Available in several options, from IP Beads alone, to complete IP/Western blot kits from goat, mouse, rabbit or sheep. 


TrueBlot® Immunoprecipitation and Western Blot Kit for DYKDDDDK (FLAG®) Epitope Tag

Substrates for Western blotting



Chemiluminescent Substrates


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.


WB9    WB10

Chemiluminescent FemtoMax™ Super Sensitive HRP Substrate




Chromogenic Substrates


Chromogenic 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 Substrates:  


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 sensitivity. 


Browse all our substrates 




Western blotting Kits


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 Control Antibodies for Western blotting 

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.


alpha-Tubulin Antibody


Popular Loading Control Antibodies for Western blotting:




 Western Blot Stripping Buffer


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. 



Blot A was first probed with Rabbit Anti-STATS (600-401-A44) then incubated with Revitablot and a competitor's product for 20 minutes at room temperature. Blot B (Blot A reprobed) with Rabbit anti-tubulin (600-401-880).


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