PhosDecor™ Fluorescent Phosphoprotein In-Gel Detection Kit

Quick & Selective Identification of Phosphorylated Proteins
Phosphorylation is an important covalent post-translational modification (PTM) in cell signaling pathways. It is estimated there are more than 100,000 sites of phosphorylation in the human proteome. Phosphorylation of a single protein can alter an entire signal transduction pathway, making each phosphorylation event potentially significant. Traditional identification of phosphorylated protein involves the use of radioisotopes or antibodies, both of which have significant costs and limitations.

• Complete in as little as 4 hours - provides quick identification of protein phosphorylation
• Gels may be dual stained for total protein - measure variations in gel loading and identify non-phosphorylation sites
• Linear detection of phosphoproteins over a wide range - allows for qualitative comparisons
• Compatible with downstream analyses such as in-gel trypsin digestion and MALDI-MS analysis - allows identification of specific phosphorylation sites within the protein
• A complete solution kit - saves time and reduces waste

Sigma now provides a quick and selective identification method for detection of phosphorylated proteins directly in SDS-polyacrylamide gels with the PhosDecor Fluorescent Phosphoprotein In-Gel Detection Kit, (PDECOR). This technology is particularly useful in screening for phosphoproteins in complex protein samples. The kit includes sufficient reagents for staining five mini-gels (8 cm x 8 cm x 1 mm). It contains stain, fixing solution, destain solution, and control.

Procedure

PhosDecor™ Fluorescent Phosphoprotein In-Gel Detection Kit Procedure (click on image or above link to view procedure in its entirety.)

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Control Proteins

The PhosDecor Control contains six proteins, two of which are phosphorylated, providing a set of positive and negative controls for use with PhosDecor Fluorescent Phosphoprotein In-Gel Detection Kit. Protein MW (kDa) Potential Phosphorylation Sites β-galactosidase 116 None Phosphorylase b 97 None Albumin 66 None Ovalbumin 45 2 β-casein 30 5 Lysozyme 15 None

A: PhosDecor Control stained with PhosDecor Stain B: PhosDecor Control dual stained with EZBlue™ Gel Staining Reagent

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Linear Detection Over a Wide Range

Staining of β-casein ranging from 3.13 ng to 200 ng using a UV transilluminator.

PhosDecor allows for qualitative comparisons in phosphoproteins with comparable phosphorylation states due to a uniform signal response to the presence of phospho groups present.

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Downstream MALDI-MS Analysis

Following PhosDecor staining, protein spots or bands can be digested using the Trypsin Profile IGD Kit (Catalog Number PP0100) and prepared for downstream MALDI-MS analysis. For accurate detection of phosphorylated peptides, digested proteins may need to be enriched prior to mass spectrometry (i.e., PhosphoProfile™ I Phosphopeptide Enrichment Kit, Catalog Number PP0410).

MALDI-MS spectra of ovalbumin stained with PhosDecor Stain.

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