The Smart Protein Layers (SPL) technology for 1D gels and Western Blots includes the addition of a bi-fluorescent standard (Smartalyzer) to every protein sample. For 1D gels and Western blots this multi-functional standard offers multiple benefits.

The Smartalyzer allows you to precisely analyze protein expression on gels and blots: lane-to-lane, target-to-protein, gel-to-gel and blot-to-blot. The precise normalization, standardization and quantification of proteins and/ or antibody signals can be performed even when unequal amounts of protein per lane have been loaded.

Features for 1D Gels

• Stain-free protein visualization, sensitivity 0.1 – 1.0 ng
• Loading by volume with exact quantification
• Normalization of unequal sample loading
• Rapid gel imaging within glass cassette
• In-gel normalization and quantification (lane to lane)
• Gel-to-gel normalization and quantification (lane to lane)
• Excision of bands in visible light

Features for Western blots

• Transfer control
• Sensitive target protein detection (5  – 10 pg)
• Signal stability over months
• In-blot normalization and quantification (lane-to-lane)
• Blot-to-blot signal normalization and quantification
• No interference of antibody recognition
• Compatible with mass spectrometry
• Step-by-step controls

 

Using the Smartalyzer for normalization, standardization and quantification

Normalization of total protein is required when for instance protein amount was not determined before separation, incorrect protein determination occurred or sample was lost when pipetted into the well.

The bi-fluorescent Smartalyzers provide signal normalization of total protein for every lane within the gel or blot. The band value for the Smartalyzer of one lane (e.g. the first lane carrying a protein sample) is set as a reference and the band volume of the Smartalyzer of lane 2 (or lane X) is divided by this reference. The raw volume of each lane is divided by the corresponding normalization factor (see Fig.1).

Gel-to-gel and blot-to-blot analysis can be performed by using a bi-/ tri-fluorescent Gel-to-Gel Smartalyzer.

Application of the Smartalyzer

To generate the sample-dependent standard, a fluorescent low-molecular weight polypeptide (Smartalyzer) is added to each protein sample. During a short and robust chemical reaction, both the sample and the Smartalyzer are labeled with a second fluorescent compound. After this  procedure, the protein of the sample now emits a strong fluorescence (color of the Smart Label, e.g.  red fluorescence), the Smartalyzer emits its basic fluorescence (e.g. blue) and the fluorescence of the total protein (color of the Smart Label e.g. red).