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Affinity Chromatography of Phosphorylated Proteins 287
Table 1
Typical Yields of Enriched Phosphoprotein From Various Cell Lines
Cell line
Loaded
(mg)
Non-adsorbed
(mg)
Washes
(mg)
Eluate
(mg)
Eluate,
%of
loaded
HEK 293 2.5 1.9 0.23 0.41 16
Jurkat 3.3 2.4 0.30 0.52 16
Cos-7 3.1 2.4 0.26 0.47 15
NIH 3T3 2.7 1.9 0.21 0.45 17
HeLa 3.4 2.5 0.24 0.46 14
Fig. 1. Western blot data for three phosphoproteins from HEK 293 enriched using
PMAC Phosphoproteins Enrichment Kit (Cat. no. 635624) according to the protocol
on page 288. Fractions from PMAC chromatography were run on SDS gel, transferred
to PVDF membrane, and stained with phospho-peptide specific antibodies for the three
proteins.
MW-Marker
Lane 1: Original Sample (total protein loaded on the column)
Lane 2: Flow through
Lane 3: Washes
Lane 4: Eluate
Western blot data for phosphoproteins from HEK 293 enriched using phosphoprotein
resin and buffers given in protocol for running sample on phosphoprotein column.
Samples from the column were analyzed by Western blotting using phosphoproteinspecific
antibodies. Phosphorylated proteins were clearly detected in the eluate
fraction.