Western Blot

Colorimetric Western Blot

A colorimetric western blot method uses an enzyme-conjugated secondary antibody and a chromogenic substrate for detection.

Advantages:

• Require no special equipment

Disadvantages:

• Sensitivity is limited (pg range)
• Blots cannot be stripped and reprobed to investigate additional targets

Chemiluminescent Western Blot

A chemiluminescent western blot uses an enzyme-conjugated secondary antibody and a luminescent substrate. Results are detected using x-ray film and darkroom equipment, or a digital imaging system.

Advantages:

• Sensitivity (fg levels)
• Blots can be stripped and re-probed

Disadvantages:

• Results can be non-linear
• Signal has a short lifetime
• Equipment required for detection can be expensive and space-consuming

Fluorescent Western Blot

A fluorescent western blot uses a secondary antibody conjugated to a fluorophore, so no substrate is required. A fluorescence imager is required to detect results.

Advantages:

• Greater dynamic range and better linearity than colorimetric or luminescent methods
• Can multiplex using different fluorophores

Disadvantages:

• Can be less sensitive than chemiluminescence
• Requires a dedicated fluorescence imaging system

ScanLater Western Blot Detection System

With the ScanLater^™ Western Blot Detection System, secondary antibody is conjugated to a time-resolved fluorophore, combining advantages of both fluorescent and chemiluminescent methods.

• No substrate required for detection
• Signal stability of months or more
• Sensitivity at sub-picogram levels
• Wide dynamic range

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Detection and quantitation of protein with ScanLater Western Blot Detection System

Protein detection is important for pharmaceutical and clinical research today, and western blots are among the most common methods employed for this purpose. Various techniques are used to detect proteins on western blot membranes including fluorescence and chemiluminescence. However, each technique has its limitations, and there is a continuing need to improve quantitation, accuracy, and dynamic range.

This application note demonstrates extended dynamic range due to reduced background as well as stability of detection over time and number of reads. In addition, a comparison of Scanlater Western Blot System to a chemiluminescence method demonstrates improved sensitivity.

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Validate CRISPR-Edited Cells using Imaging and Western Blot Detection

Genome editing has been widely used to study gene expression and protein function, but many of these methods are labor intensive and inaccurate1 . The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has become a very popular tool for editing genes due to its high accuracy and ease of use2.

Here, we demonstrate how the SpectraMax i3x Multi-Mode Microplate Reader and ScanLater Western Blot Detection System can be used to validate CRISPR/Cas9 genome editing.

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Wells to Westerns: Investigating the cellular heat shock response

Investigating a cellular response can involve multiple approaches to gathering information—imaging, cell-based viability and proliferation assays, western blots to look at changes in protein expression, and more. Often multiple instrument platforms are needed to glean the necessary results, and several software packages may need to be learned in the process.

In this application note we show how data for several related cellular parameters was collected using a single instrument, the SpectraMax® i3 Multi-Mode Detection Platform.

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Estimation of Protein Molecular Weights with ScanLater Western Blot Protein Ladder

The ScanLater™ Western Blot Protein Ladder is an essential component of the ScanLater™ Western Blot Detection System. The primary applications of this protein ladder include the estimation of protein molecular weight, the visualization of gel electrophoresis, and the evaluation of the transfer process from gel to blot.

Learn how users can follow the electrophoresis and blotting workflow that is optimized for their application.

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Protein Detection Based on Europium Labeled Proteins

Western Blot Protein Detection and Quantitation Based on Europium Labeled Proteins using a Plate Reader

Here we report a novel system for western blot analysis that is incorporated into a SpectraMax® multi-mode microplate reader. Membranes are probed with europium labeled secondary antibodies or streptavidin that label the protein of interest. Europium (Eu) has a long fluorescence lifetime, on the order of 1 msec, and detection is done with time resolved fluorescence (TRF) detection mode, which significantly reduces background from auto-fluorescence or other sources of short lifetime emissions.