Download Application Note #2 - GE Healthcare Life Sciences

A M E R S H A M
B I O S C I E N C E S
Phosphoamino Acid Analysis by
Thin Layer Electrophoresis
Contributed by Charles W. Mahoney, Noriyuki Nakanishi, Motoaki Ohashi
Tanabe Pharmaceutical Company Ltd., Toda, Japan
Application Note #2
Multiphor® II
A) Ninhydrin
B) Autoradiograph
Keywords: Phosphoprotein; p53
Introduction
Proteins are often phosphorylated in vivo on Ser, Thr, and/or
Tyr residues. As this phosphorylation often serves a regulatory role in protein and cellular function, identification of the
phosphorylated amino acid(s) in a particular protein is
important in signal transduction research. This is commonly
done by hydrolyzing the 32P-labeled phosphoprotein and
separating the hydrolysate by thin layer electrophoresis on
cellulose TLC plates. The phosphorylated amino acids are
identified by comparing the mobility of phosphoamino acids
visualized by autoradiography to the mobility of phosphoamino acid standards visualized with ninhydrin [1-6].
P-Ser
P-Thr
P-Tyr
BPB
Here we describe a protocol for rapid, reproducible phosphoamino acid analysis by semi-dry thin layer electrophoresis in
the Multiphor II electrophoresis unit [5].
O rigin
Results
This technique was applied in order to identify the phosphorylated amino acid(s) in immunoprecipitated p53 that had
been labeled with 32P phosphate in vivo. The results clearly
indicate the presence of phosphoserine alone (Figure 1).
This technique has proved to be rapid and reproducible, and
it is performed on a widely available flatbed electrophoresis
apparatus.
Fig. 1. Phosphoamino acid analysis of 32P-labeled p53 by semi-dry cellulose thin-layer electrophoresis. A) Photograph of the TLC plate stained
with ninhydrin. B) Autoradiograph of the TLC plate. Positions of phosphoserine, phosphothreonine, phosphotyrosine and the bromophenol
blue marker dye are as indicated. Figure used with permission of
Academic Press [5].
Methods
SDS-PAGE and electrotransfer
A sample containing 32P-labeled protein was separated by
standard Laemmli SDS-PAGE and blotted onto a polyvinylidine difluoride (PVDF) membrane (Immobilon P, Millipore).
The SDS-PAGE can be done using a SE 600 or
SE 400 gel electrophoresis unit. The electrotransfer can be
done in a semi-dry mode on a TE 70 or TE 77
SemiPhor™ semi-dry transfer unit, or in tank mode in a
TE 62 or TE 42 Transphor™ tank transfer unit.
The equipment manufacturer’s instructions should be
followed for the SDS-PAGE and electrotransfer.
80-6413-07
Rev A / 11-97
Semi-dry cellulose thin layer
electrophoresis
Sample preparation
Solutions
6 N HCl
(6 N Hydrochloric acid, 10 ml)
Concentrated HCl (~12 N)
Distilled water
Materials
Glass-backed cellulose TLC plates, 20 x 20 cm, or 10 x 20 cm
are available from Merck (Germany), catalog number
1.05716.0000. These plates are distributed in North America
by:
5 ml
5 ml
30% methanol, 0.1 N HCl
(30% methanol, 0.1 N Hydrochloric acid, 10 ml)
Methanol
Concentrated HCl (~12 N)
Distilled water
EM Separations Technology
480 Democrat Rd.
PO Box 70
Gibbstown, NJ 08027
Tel: 609-224-0742
Fax: 609-423-4389
3 ml
83 µl
to10 ml
Phosphoamino Acid Standards
(0.5% phosphoserine, 0.5% phosphothreonine, 0.5% phosphotyrosine, 1 ml)
O-Phospho-L-serine (FW 185.1)
O-Phospho-L-threonine (FW 199.1)
O-Phospho-L-tyrosine (FW 261.2)
Distilled water
For a list of other international distributors, contact:
Merck KGaA
64271 Darmstadt
Frankfurter Straße 250
Tel: 49-6151-72-0
Fax: 49-6151-72-2000
5 mg
5 mg
5 mg
1 ml
Solutions
Bromophenol Blue Tracking Dye
(0.1% bromophenol blue, 1 ml)
Bromophenol blue (FW 691.9)
Distilled water
Electrophoresis Buffer
(5% (v/v) acetic acid, 0.5% (v/v) pyridine, pH 3.5, 100 ml)
1 mg
1 ml
Acetic acid (glacial)
Pyridine
Distilled water
Following transfer of the 32P-labeled proteins to the PVDF
membrane, the membrane was marked with luminescent or
radioactive markers for orientation, covered with plastic
wrap and exposed to x-ray film with the protein side towards
the film (the length of the exposure will vary with the abundance of the protein and its phosphate content). After
developing the autoradiograph, the membrane was aligned
with the x-ray film on a light box. A razor blade was used to
excise the area of PVDF membrane corresponding to the
32
P-labeled protein band of interest.
5 ml
500 µl
94.5 ml
The MultiTemp® III thermostatic circulator was connected to
the Multiphor II electrophoresis unit and pre-cooled to 16 °C.
The glass backed cellulose TLC plates were marked with a
blunt soft pencil at 3 and 9 cm for the origin and migration
point for the bromophenol blue tracking dye, respectively.
Phosphoamino Acid Standards (1 µ1, ~25 nmol of each phosphoamino acid) were applied to each lane at the origin using
a glass capillary or 1 µl pipetter, drying between each application with a cool hair dryer. Hydrolyzed 32P-labeled protein
was applied in a similar manner to each spot at the same
origin pre-spotted with Phosphoamino Acid Standards.
Bromophenol Tracking Dye (1 µl) was applied to a separate
lane. The TLC plate was gently sprayed with Electrophoresis
Buffer and placed in the center of and parallel to the long
axis of the Multiphor II cooling plate with the origin towards
the cathodic (-) end. Amersham Biosciences CleanGel™ electrode
strips were cut to the width of the TLC plate (5 x 10.5 cm or
5 x 20.5 cm) and pre-soaked with Electrophoresis Buffer
The immobilized 32P-labeled protein was hydrolyzed by
incubating the membrane slice in 200 µl 6 N HCl for 2 h at
110 °C in a securely capped 1.5 ml polypropylene microcentrifuge tube. The amino acids were eluted from the
membrane with 30% methanol, 0.1 HCl (3 changes, 200 µl
each). The 6 N HCl hydrolysate and the three eluates were
pooled and dried in a speed-vac, or by lyophilization.
The sample was resuspended in 10 µl of distilled water.
2
Glass Plates
Electrode Plate
Detection
Cathod
Solutions
Ninhydrin Solution
(0.2% (w/v) ninhydrin in ethanol, 10 ml)
Ninhydrin
20 mg
Ethanol (reagent grade) 10 ml
Anode
TLC Plate
The TLC plate was removed from the Multiphor II unit and
dried for 10 min with a hot hair drier. The plate was sprayed
with Ninhydrin Solution and blown with a hot hair drier for
10 min to visualize the phosphoamino acid standards. The
TLC plate was exposed to X-ray film to determine the position of the 32P phosphoamino acids. Spotting radioactive or
luminescent markers onto the dried TLC plate helped in
aligning the autoradiogram to the TLC plate.
CleanGel
Electrode Strip
Fig. 2. Schematic of the Multiphor II flatbed electrophoresis unit set up
for phosphoamino acid analysis. (See text for details). Redrawn from [5].
(10 ml or 20 ml depending, respectively, on the length of the
CleanGel electrode strips). The wet strips were overlapped
2 cm onto each end of the TLC plate and three glass plates
the same size and thickness of the TLC plate were placed on
top of the TLC plate with the wicks in place. TLC plates
from which the cellulose layer has been scraped off can be
used. (The weight of the glass plates was found to be critical
for maintaining good electrode strip to TLC plate contact
and the proper humidity). The interelectrode distance was set
to 24 cm and the electrode plate was placed over the TLC
plate, glass plates and buffer-soaked electrode strips so that
the electrodes rested on the electrode strips with the cathode
towards the origin (see Figure 2). The lid was placed on the
Multiphor II and connected to a high voltage power supply
set for 1000 V, with current and power limits set at 30 mA
and 30 W, respectively. Electrophoresis was run at constant
voltage for 40-50 min and terminated when the
Bromophenol Blue Tracking Dye had migrated 6 cm.
Note in Proof
It has been found that use of the following alternative electrophoresis buffer results in greater separation among the
three phosphoamino acids (data not shown) [6].
Electrophoresis Buffer
(5.93% (v/v) acetic acid, 0.73% (v/v) formic acid,
0.33% (v/v) pyridine, 0.33 mM EDTA, pH 2.5, 100 ml)
Acetic acid (glacial)
Formic acid (88%)
Pyridine
0.5 M EDTA, disodium salt
Distilled water
3
5.93 ml
830 µl
330 µl
66 µl
92.8 ml
References
1. Cooper, J.A, Sefton, B.M. and Hunter, T. Detection and quantification of phosphotyrosine in proteins. Meth. Enzymol. 99, 387-402
(1983).
2. Kamps, M.P. and Sefton, B.M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino
acids in gel-fractionated proteins. Anal. Biochem. 176, 22-27 (1989).
3. van der Geer, P., Luo, K., Sefton, B.M. and Hunter, T.
Phosphopeptide mapping and phosphoamino acid analysis on cellulose thin-layer plates. Protein Phosphorylation - A Practical
Approach. Oxford University Press Inc., NY (1993).
4. van der Geer, P and Hunter, T. Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on
thin-layer cellulose plates. Electrophoresis 15, 544-554 (1994).
5. Mahoney, C.W., Nakanishi, N. and Ohashi, M. Phosphoamino acid
analysis by semidry electrophoresis on cellulose thin-layer plates
using the Amersham Biosciences or Atto flatbed apparatus.
Anal. Biochem. 238, 96-98 (1996).
6. Jelinek, T. and Weber, M.J. Optimization of the resolution of phospho-amino acids by one-dimensional thin-layer electrophoresis.
BioTechniques 15,629-630 (1993).
Ordering Information
Code No.
80-6171-58
80-6154-86
80-6210-34
80-6211-86
80-6205-98
80-6209-58
18-1018-06
18-1102-77
18-1102-78
19-3500-00
18-1035-33
17-1324-01
17-1329-01
Item Description
SE 600 Dual Vertical Gel Unit
SE 400 Sturdier Vertical Gel Unit
TE 70 SemiPhor Semi-Dry Transfer Unit
TE 77 SemiPhor Semi-Dry Transfer Unit
TE 42 Transphor Tank Transfer Unit
TE 62 Transphor II Tank Transfer Unit
Multiphor II Electrophoresis Unit
MultiTemp® III Refrigerated Bath Circulator, 100-120 V
MultiTemp III Refrigerated Bath Circulator, 200-220 V
EPS 3500 Power Supply
CleanGel electrode strips, package of 12
EDTA, disodium salt, 100 g
Bromophenol Blue, 10 g