Immunoblotting Assays
Immunoblotting Assays - Related Videos
16:01
Immunoblot Analysis
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Cited by 21 •
2008
Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.
06:00
SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting
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2025
This video demonstrates the in vitro method for SUMOylation of substrate proteins using a sequential enzyme cascade. Further, the SUMOylated status of the protein is identified using the electrophoresis and immunoblotting technique.
09:32
Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting
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2025
This article describes a protocol for analyzing proteins from Xenopus oocytes and embryos by immunoblotting. Collection steps are described, followed by steps corresponding to sample processing, SDS-PAGE, transfer, antibody staining, and imaging. The protocol emphasizes studying translational regulatory protein complexes with endogenous antibodies and antibodies against protein affinity tags.
08:08
Co-Immunoprecipitation and Pull-Down Assays
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2023
Co-immunoprecipitation (CoIP) and pull-down assays are closely related methods to identify stable protein-protein interactions. These methods are related to immunoprecipitation, a method for separating a target protein bound to an antibody from unbound proteins. In CoIP, an antibody-bound protein is itself bound to another protein that does not bind with the antibody, this is followed by a separation process that preserves the protein-protein complex. The difference in pull-down assays is that...
08:57
Native Polyacrylamide Gel Electrophoresis Immunoblot Analysis of Endogenous IRF5 Dimerization
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Cited by 1 •
2019
A native Western blot method for analyzing endogenous interferon regulatory factor 5 dimerization in the CAL-1 plasmacytoid dendritic cell line is described. This protocol can be applied to other cell lines as well.
FAQs
Gel electrophoresis separates proteins according to size before they are transferred to a membrane. This separation places proteins in distinguishable positions, so antibody binding can be associated with a particular protein population rather than with the entire complex sample. The resulting pattern helps researchers characterize targets and compare protein-related changes across biological samples.
The primary antibody selectively binds the protein of interest, while the labeled secondary antibody binds the primary antibody and supports signal detection. This two-antibody arrangement connects molecular recognition with an observable result after protein separation. Researchers can therefore determine whether the target is present and assess its relative abundance within the analyzed sample.
Immunoblotting can provide information about protein processing, localization, and post-translational changes in addition to expression. These dimensions help researchers examine how proteins are altered or distributed during biological processes, including molecular signaling. Consequently, the assay can contribute to a more detailed view of protein behavior rather than only indicating whether a protein is produced.
Immunoblotting provides a complementary validation approach for results obtained with other protein-analysis methods. Because it combines size-based separation with selective antibody recognition, it can support the interpretation of findings about a particular protein in a complex sample. This role is especially useful when researchers need independent evidence for changes in expression or protein characteristics.
Applications span cell biology, genetics, microbiology, and disease studies. In these fields, researchers can investigate whether particular proteins are expressed, compare their relative abundance, and examine processing or post-translational changes. The same general approach therefore supports questions about cellular behavior, genetic effects, microbial biology, and disease-associated protein patterns.
A detectable signal indicates that the antibody-based detection system has identified the target protein in the analyzed sample. When researchers compare signals across samples, the assay can also provide information about relative target abundance. Interpreting the signal alongside protein separation may further support conclusions about processing or other protein characteristics relevant to the biological question.