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Th Thermo Scientific Pierce
Phosphoprotein Enrichment Kit

Obtain high phosphoprotein yields with an easy-to-use spin format

FT W E L
Phospho-MAPK
T202/Y204

Phospho-Akt S473

Phospho-Src Y527

Phosphorylation is one of the most frequently occurring post-
translational modifications in proteins and it regulates nearly
all intracellular biological events, including signal transduction,
protein-protein interaction, protein stability, protein localization,
apoptosis and cell cycle control. Detecting changes in protein
phosphorylation can be a difficult task because of the transient,
labile state of the phosphate group. Furthermore, low phospho-
protein abundance and poorly developed phospho-specific
antibodies also contribute to difficulties in phosphoprotein
detection. Recent advances in mass spectrometry technology
combined with phosphoprotein enrichment using immobilized
metal affinity chromatography (IMAC) have resulted in greater
resolution of the phosphoproteome.
The Thermo Scientific Pierce Phosphoprotein Enrichment Kit
is designed for the efficient enrichment of phosphorylated
proteins derived from mammalian cells and tissues. The propri-
Cytochrome C
p15Ink4b
Figure 1. Enrich complex biological samples for phosphorylated proteins
with high specificity. Western blotting analysis reveals a stronger signal for
the elution fraction (E) bands (processed sample) than the total cell extract
(L) bands (unprocessed sample) for all phosphoproteins tested. Furthermore,
the non-phosphorylated proteins cytochrome c and p15Ink4b are negative
controls and demonstrate the specificity of the kit. Total cell extract (2 µg)
from serum-starved NIH 3T3 cells was used for each enrichment procedure.
Ten µg of total protein was loaded in each lane and Western blotting was
performed using antibodies that detect site-specific phosphorylation.
FT: flow-through and W: wash.
L FT W E
Phospho-PTEN S380
Phospho-Rb S795
Cytochrome C

etary resin and buffer composition produces superior yields

with negligible nonspecific binding. The kit is offered in a
convenient spin format and is compatible with downstream
applications, including Western blotting, mass spec,
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
and protein arrays. The entire enrichment process, from cell
lysis through concentration, can be completed in less than
two hours using the Pierce Phosphoprotein Enrichment Kit.
Highlights:
Figure 2. Efficient phosphoprotein enrichment and high specificity from
mouse liver extract. Western blot analysis reveals a stronger signal for the
elution fraction (E) bands (processed sample) than for the same amount of
total cell extract (L) bands (unprocessed sample) for both phosphoproteins
tested. Furthermore, the non-phosphorylated protein cytochrome c is a
negative control and demonstrates the specificity of the kit. Approximately
2 mg of homogenized mouse liver extract was loaded onto a
Phosphoprotein Enrichment Column. Concentrated flow-through (FT),
wash (W), and elution (E) fractions were resolved by SDS-PAGE. Ten µg of
total protein was loaded in each lane and Western blotting was performed
using antibodies that detect site-specific phosphorylation.

• Specific – low nonspecific protein contamination from Table 1. The Thermo Scientific Pierce Phosphoprotein Enrichment Kit
complex biological samples such as cell culture lysate and provides higher phosphoprotein yields in less time than other kits.
mouse tissue extract
Phosphoprotein Enrichment
• Fast – spin format enables efficient enrichment in less than Kit Yield (µg)* Time (Hours)
two hours Our Phosphoprotein 300 1.5
• Superior yield – achieves higher yields than other Enrichment Kit
commercially available kits (Table 1) Supplier Q Kit 88 4.5
• Convenient – complete kit contains pre-dispensed spin Supplier I Kit 52† 3.5
columns, buffers, reagents and ultrafiltration columns Supplier C Kit 160 3
• Compatible – downstream applications include mass Supplier E Kit Too dilute to determine 5
* From 2 mg total protein; † Based on maximum 1 mg load per manufacturer’s
spectrometry, Western blotting and 2D-PAGE protocol
 Thermo Scientific Pierce Phosphoprotein Enrichment Kit

Ordering Information:

Product # Description Pkg. Size

90003 Pierce Phosphoprotein Enrichment Kit Kit
Includes: Phosphoprotein Enrichment Column Resin Bed (1 ml) 10 ea.
Lysis/Binding/Wash Buffer 325 ml
Elution Buffer 60 ml
CHAPS 1g
White Column Caps
Pierce Protein Concentrators 7 ml/9K MWCO 10 devices

Related Products:

Product # Description Pkg. Size

89804 Glycoprotein Isolation Kit, ConA Kit
Sufficient reagents to isolate glycoproteins with strong affinity for ConA
from 10 samples of up to 640 µl each (1-1.5 mg total protein)
Kit contains: ConA Lectin Resin 1.1 ml settled resin supplied as a 50% slurry
Binding/Wash Buffer 6.5 ml of a 5X stock solution
Elution Buffer 5 ml
Column Accessory Pack 10 spin columns with bottom caps and 20 collection tubes
89805 Glycoprotein Isolation Kit, WGA Kit
Sufficient reagents to isolate glycoproteins with strong affinity for WGA
from 10 samples of up to 640 µl each (1-1.5 mg total protein)
Kit contains: WGA Lectin Resin 1.1 ml settled resin supplied as a 50% slurry
Binding/Wash Buffer 6.5 ml of a 5X stock solution
Elution Buffer 5 ml
Column Accessory Pack 10 spin columns with bottom caps and 20 collection tubes
89853 Phosphopeptide Isolation Kit Kit
Sufficient for analyzing 30 protein digestions.
Kit contains: Mini-spin columns with SwellGel® Gallium-Chelated Disc 30 ea.
89899 Ubiquitin Enrichment Kit Kit
Contains sufficient materials for enriching up to 15 lysate samples
containing ~0.15 mg total protein per sample.
Kit contains: Pack 1
Polyubiquitin Positive Control (1,000 X), 2 mg/ml 50 µl
Anti-Ubiquitin Antibody, rabbit anti-serum 50 µl
Pack 2
Polyubiquitin Affinity Resin 300 µl
BupH™ Tris Buffered Saline Pack 1 pack
Spin Columns and Accessories 18 columns
78420 Halt™ Phosphatase Inhibitor Cocktail 1 ml
A mixture of four phosphatase inhibitors including: sodium fluoride,
sodium orthovanadate, sodium pyrophosphate and beta-glycerophosphate.
78430 Halt Protease Inhibitor Single-Use Cocktail (100X) 24 x 100 µl
Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.
Protease inhibitors prepared in DMSO solution.
Kit contains: Protease Inhibitor Cocktail, 100 µl microtubes 24
0.5 M EDTA Solution 2.5 ml
78425 Halt Protease Inhibitor Single-Use Cocktail, EDTA-free (100X) 24 x 100 µl
Each 100 µl microtube contains sufficient cocktail to treat 10 ml of lysate.
Protease inhibitors prepared in DMSO solution.
Kit contains: Protease Inhibitor Cocktail, 100 µl microtubes 24
89806 Protein Stabilizing Cocktail 10 ml
4X Concentrated Solution

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