Dot Immunoblot
Dot Immunoblot - Related Videos
16:01
Immunoblot Analysis
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Cited by 25 •
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.
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.
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.
10:16
Production and Targeting of Monovalent Quantum Dots
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Cited by 2 •
2014
We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.
04:39
Quantitative Dot Blot to Estimate Target Proteins in a Tissue Lysate
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2025
This video demonstrates a quantitative dot blot, or QDB, technique for the absolute quantification of proteins of interest in a tissue sample. The proteins in the sample are immobilized on the nitrocellulose membrane inside the wells of a QDB plate. Utilizing target protein-specific antibodies labeled with a peroxidase enzyme, a chemiluminescent substrate is oxidized to produce light, which is measured to determine the protein concentration of the sample.
FAQs
Blocking reduces nonspecific binding at the membrane before antibody detection begins. This preserves the distinction between signal produced through recognition of the target by the primary antibody and background interactions that could obscure it. In practice, effective blocking supports clearer interpretation of whether a spot reflects the protein or antigen of interest.
The primary antibody provides target recognition by binding the protein or antigen deposited on the membrane. A labeled secondary antibody then functions in the detection system after primary-antibody recognition and generates the visible or measurable signal. This arrangement translates target presence into an observable spot without requiring the sample’s proteins to be separated first.
Signal presence provides qualitative evidence that the target protein or antigen is present, whereas signal intensity can support a semi-quantitative comparison among samples. In genetics research, those comparisons can indicate differences in protein products associated with genetic variants. The result does not replace full protein separation when detailed separation is required, because the assay is designed for screening.
Researchers apply each biological sample as a discrete spot to a membrane, block nonspecific binding sites, add a primary antibody, and then use a labeled secondary antibody to produce a visible or measurable signal. Comparing the resulting spots provides the readout. This workflow supports parallel handling of many samples while avoiding a protein-separation step.
It places samples in a common membrane-based detection workflow, so researchers can compare whether the target protein is detected and how strongly it appears across variants. The resulting presence or intensity differences provide qualitative or semi-quantitative evidence about protein products associated with those genetic backgrounds. This makes the assay useful for screening expression patterns rather than resolving proteins individually.
A sample containing a suspected recombinant product can be spotted onto the membrane and tested with antibodies directed toward the relevant target. Detection supplies evidence that the protein product is present, while signal strength permits a semi-quantitative comparison across samples. Because many spots can be screened without full protein separation, the method is suited to efficient production checks.