Phosphoprotein Enrich-

EzWay Phos-Pro Enrichement Kit

Catalog Number	K07920
Size	10T
Type	Kit
Datasheet

Background

Protein phosphorylation is one of the most frequently occurred posttranslational modifications and plays a critical role in cellular regulatory events. Most cellular processes are in fact regulated by the reversible phosphorylation of proteins on serine, threonine and tyrosine residues. In fact, phosphorylation of proteins plays a key role in oncogenesis, cell signaling, apoptosis and immune disorders1.
Despite the importance and widespread occurrence of this modification, profiling of phosphoproteins in cells is still a challenge, due to the low copy of phosphorylated proteins in cell and the relative amount of phosphoproteins compared to unphosphorylated proteins.

Ladiolabeling by 32P labeling is frequently used conventional method for investigation of phosphoprotein profile in conjunction with 2-DE or 1-D gel electrophoresis and autoladiogram. Alternatively, western blot analysis probed by phosphoprotein-specific antibody is also used for this purpose.
Mass spectrometry has been shown to be a reliable and routine tool to identify proteins in a high throughput manner.
However, the identification of phosphorylation by mass spectrometry is not a trivial matter and to this day is not routine also due to the low copy of phosphorylated proteins in cells.

EzWay™ Phos-Pro Enrichment Kit provides advanced chance in detecting protein phosphorylation in gels with non-radiolabeling method(eg. Staining with fluorescence dye) and enables quantitative comparison between cells.

EzWay™ Phos-Pro Enrichment Kit is optimized for the protein solution in denatured condition, for example, the samples prepared for 2-DE and can be adapted to native proteins. Enriched phosphorylated proteins could be detected by staining commercially available staining method using fluorescent dye2 or by probing with antibodies, specific for phosphorylated proteins.

Features

Convenient fractionation of phosphoproteins from protein mixtures such as cell lysate or body fluids
Isolation and concentration of low copy phosphorylated proteins in cells
No use of IMAC column of phosphorylated antibodies
Downstream application for 2-DE or 1-D SDS-PAGE analysis
Enriched phosphorylated proteins detection by staining with fluorescent dye or antibody probing
Specificity 100%
Selectivity >87%
Sensitivity >93% / 50ng
Yield >93%

Kit Contents

Lysis Buffer
Dilution Buffer
Phos-Pro Solution A
Phos-Pro Solution B
Dissolving Solution
Delipidation Solution

How to Use

Cell lysis and Protein extraction
Precipitation of phosphoprotein complex
Dedelipidation and recovering of phosphoprotein

Phosphoprotein Enrichment from Yeast cell Extract

CBB Staining

Phosphoprotein Staining
(ProQ Diamond)

Sup of 8
Sup of 9
Sup of 10
Sup of 11
Sup of 12
sup of 13
Yeast lysate enriched (0.4mg/ml)
Yeast lysate enriched (0.4mg/ml)
Yeast lysate enriched (0.4mg/ml)
Yeast lysate enriched (0.4mg/ml)
Yeast lysate enriched (0.4mg/ml)
Yeast lysate enriched (0.2mg/ml)
Yeast lysate enriched (0.1mg/ml)
Total cell Extract


Yeast total protein extract CBB Staining	Enriched phosphorylated protein CBB staining	Enriched phosphorylated protein ProQ Diamond Staining