Western blotting

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Applications:Western blotting

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Methods Used to Detect Proteins and Nucleic Acids Bound to Membranes

Background and protocols describing the various methods used by molecular biologists to detect samples of protein or nucleic acids bound to membranes.

Semi-Dry or Tank Electrotransfer Troubleshooting

The possible causes and potential remedies for poor or inconsistent transfer of proteins from a gel to the Western blot membrane. Optimal transfer conditions may vary depending on the molecular weight of the protein of interest.

Immunodetection

The possible causes and potential remedies for challenges encountered as a result of blocking, washing, antibody incubation, and detection/exposure of Western blots

Western Blotting: Why Are Observed and Calculated Molecular Weights Different?

A discussion of factors affecting observed vs. calculated molecular weight in gel electrophoresis and western blotting.

Antibodies in Practice: Conjugated and Secondary Antibody Use

Using conjugated antibodies and secondary antibodies in the lab.

Loading Controls for Western Blotting

Loading controls in western blotting application.

Antibody Standard Validation

Explore antibody validation, including antibody and target, species validation, lot validation, application validation, storage and handling, citations, and quality control, in detail.

An Introduction to Antibodies: Antigens, Epitopes and Antibodies

Antibodies will combine with specific antigens to generate an exclusive antibody-antigen complex. Learn about the nature of this bond, and the use of this robust and specific binding as a molecular tag for research.

Prestige Antibodies® Organelle Markers

Subcellular localization studies are important for mapping and characterizing proteins and thus for better understanding of the cellular functions of the proteins. By confocal microscopy analysis on human cell lines, spatial and temporal protein expression patterns can be visualized on

esiRNA Knockdown Efficiency Tested by Western Blotting

Quantitative and qualitative western blotting to validate knockdown by esiRNA.

Fluorescent Multiplex Detection using Antibody Atto Dye Conjugates

Immunoblotting (Western blot transfer) is a common technique in modern proteomics research.

Western Blot Analysis of Immunoprecipitation (IP-Western)

The possible causes and potential remedies for challenges encountered in the immunoprecipitation-Western blot technique, which consists of cell lysis, formation of the antibody-antigen (immune) complex, precipitation of the immune complexes, and analysis by Western blotting.

Sample Preparation & Gel Electrophoresis Troubleshooting

The possible causes and potential remedies for challenges encountered during preparation of samples for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and optimizing electrophoresis conditions.

Auto2D® Automated 2-D Gel Electrophoresis Device

The Auto2D® 2-D Electrophoresis Device fully automates difficult 2D electrophoresis methods in a quick, easy, and reproducible way. Locate difficult-to-find proteins using the Auto2D® system in less than two hours with high reproducibility.

Antibody Basics

Explore the basics of working with antibodies including technical information on structure, classes, and normal immunoglobulin ranges.

Protein Visualization

Prior to probing a membrane using precious antibodies, it is helpful to visualize the transferred proteins on the blot to ensure complete transfer and even loading. This page describes possible causes and potential remedies for challenges encountered during protein visualization.

Antibody Enhanced-Validation Strategies

Antibody enhanced validation provides additional data to help ensure antibody specificity and performance and to address antibody reproducibility crisis.

Alkaline Phosphatase

We offer a broad range of alkaline phosphatase enzymes and substrates that are optimized for conjugation to antibodies and other proteins for your specific application needs.

Electrophoresis Protein Staining Reagents Selection Guide

To meet the great diversity of protein analysis needs, Sigma offers a wide selection of protein visualization (staining) reagents. EZBlue™ and ProteoSilver™, designed specifically for proteomics, also perform impressively in traditional PAGE formats.

Antibody Generation

The inherent immune ability of an animal can be leveraged to generate antibodies that bind to specific molecules.

Anti-HA Troubleshooting

Antibodies to Tubulins, including Centrosome-Associated ε-Tubulin

Microtubules of the eukaryotic cytoskeleton are composed of a heterodimer of α- and β-tubulin. In addition to α-and β-tubulin, several other tubulins have been identified, bringing the number of distinct tubulin classes to seven.

Dot/Slot (Filtration) Blotting

The possible causes and potential remedies for challenges encountered dot/slot blotting, which uses vacuum filtration to transfer protein onto a microporous membrane.

Colorimetric Detection of Western blot using 4-Chloro-1-Naphthol Solution Protocol

This solution is used as a substrate for detecting horseradish-peroxidase conjugates in Western blotting.

Preparing Cell Lysates

This page describes common challenges encountered when lysing cells and extracting proteins prior to Western blotting. Total protein concentration must be determined for these cell lysates. Variables affecting each of these steps are outlined below, as each could affect the

Prestige Antibodies® in Breast Cancer Research

The Human Protein Atlas; Prestige Antibodies®; Polyclonals and Monoclonals; Clinical Markers; Antibodies used in Breast Cancer Research; Antibodies Against MammaPrint and Other Gene Expression Test Proteins; Antibodies Identified in the Human Protein Atlas; Finding Cancer Biomarkers, as Exemplified by RBM3

Identification of Proteins in Lipofuscin Using Antibodies within the Human Protein Atlas

Secondary Antibodies, Conjugates, and Kits

Technical Support for Secondary Antibodies, Conjugates, and Kits

Improving Reproducibility: Best Practices for Antibodies

In the midst of beeping lab timers, presentations and grant deadlines, it is easy to take for granted the quality of lab reagents.

Prestige Antibodies^® on The Cell Atlas

Visualize the spatial distribution of proteins in cells using the Cell Atlas part of the Human Protein Atlas. For each protein, there are multiple immunofluorescent staining images in a vast number of cell lines.

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