Download Protocol 1. Sample Preparation 2. Two Dimensional Polyacrylamide

Protocol
1. Sample Preparation
2. Two Dimensional Polyacrylamide Gel Electrophoresis
3. Image acquisition and Data Analysis
4. Cleveland Peptide Mapping
5. Western Blotting
6. Deblocking of Blotted Proteins
7. N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search of
Amino Acid Sequence
8. Mass Spectrometry
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1. Sample Preparation
2.1. Chemical
Lysis buffer (10ml)
Urea (9.5 M)
4.8 g
NP-40 (2%)
0.2 ml
Ampholine 2% (pH 3.5 – 10)
0.2 ml
B-mercaptoethanol(5%)
0.5 ml
PVP-40
0.5 g
Water
9.10 ml
Note. This buffer may be stored frozen at –20 o C. Do not continuously freeze and
thaw it. Use an aliquot once and discard the remainder
2.2. Method
a. Collect sample and homogenize in lysis buffer.
b. Centrifuged at 15,000 rpm for 10 min
c. Take the supernatant and centrifuge again at 15,000 g for 10 min
d. Take the supernatant and use for 2D-PAGE
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2. Two Dimensional Polyacrylamide Gel Electrophoresis
2.1. First Dimensional Isoelectric Focussing (IEF)
2.1.1 Materials.
a. 30% acrylamide stock solution
(28.38% acrylamide)
28.38 g
(1.62% BIS)
1.62 g
Make the volume to 100 ml with water
Acrylamide is a potent neurotoxin and should never be mouth pipeted.
solution is light sensitive and should be stored in the dark at 4 o C.
b. Anode electrode solution
0.02N H 3 PO 4
(1M stock: 6.364 ml phosphoric acid to 100 ml water)
c. Cathode electrode solution
0.02 N NaOH
(1M stock: 4 g Sodium Hydroxide to 100ml water)
d. SDS Sample buffer ( pH 6.8)
0.06 M Tris HCl
3.785 g
2 % SDS
10 g
5% B-mercaptoethanol
25 ml
10% Glycerol
50 ml
Make the volume up to 500 ml with water (MQ)
Acrylamide
2.1.2 Method
a. Mark 2cm from the top of clean, dry 13 cm long and 3 mm diameter, 1d gel tubes to
indicate the desired height of the gel. Place a rubber band around them so that they
form a tight bundle. Hold the bundle vertically on a flat surface. Seal the bottom
with two layers of parafilm
b. Prepare the solution
Urea
4.8 g
30% acrylamide stock
1.16 ml
NP-40
2 ml
Water
2.84 ml
Ampholines (pH5-7)
0.25 ml
Ampholines (pH 3.5 –10)
0.25 ml
10% APS
15 µl
TEMED
10 µl
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Note. Dissolve the urea by swirling in water bath whose temperature should not
higher than 60 o C
c. Using a long narrow guage hypodermic needle, fill the gel tubes with the solution of
step no 3 to the mark.
d. Overlay the gel mix with 20 µl of water and allow it to polymerize it for one hour
e. Remove the para-film from the bottom of the tubes and water is removed from the
top of the gel.
f. Seat the tubes in the holes of the upper buffer reservoir of the tube cell. Plug any
unused hole with rubber stopper
g. The lower chamber is filled with 0.02 N Phosphoric acid . The samples are loaded in
a volume 5 – 100 µl and overlaid with 20 µl of 1/2 lysis buffer. The upper chamber
is filled with 0.02 N Sodium Hydroxide.
h. Run the current as follows
200 V for 30 min
400 V for 16-18 h
600 V for 1 h
i. Extrude the gel from the tubes using the water pressure from a syringe. Wash each
gel in 5 ml SDS sample buffer for 15 minutes. Change the buffer and wash it again
for 15 minutes. At this point the gel can be stored at –20 o C for several week or used
immediately.
2.2. First Dimensional Immobilized pH Gradient (IPG)
a. Seat the tubes in the holes of the upper buffer reservoir of the tube cell. Plug any
unused hole with rubber stopper
b. The lower chamber is filled with 0.02 N Sodium Hydroxide.
c. The water is removed from the top of the gel. The samples are loaded in a volume 5
– 100 µl with a syringe and overlaid with 20 µl of 1/2 lysis buffer. The upper
chamber is filled with 0.02 N Phosphoric acid. Connect positive terminal with lower
chamber (acidic) and negative terminal to upper chamber (basic). Connect positive
terminal to lower chamber (basic) and negative terminal to upper chamber (acidic).
Run the current as follow
400 V for 1 h
1,000 V for 16 h
2,000 V for 1 h
d. Remove the tubes and force the gels out onto a parafilm. Wash each gel in 5ml SDS
sample buffer for 15 minutes. Change the buffer and wash it again for 15 minutes. At
this point the gel can be stored at –20o C for several week or used immediately
2.3. Second Dimension with IEF and IPG
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2.3.1. Materials
a. Separating gel buffer (pH 8.8)
Tris HCl (1M)
12.11 g
SDS (0.27 %)
0.27 g
Make the volume to 100 ml with water (MQ)
b. Stacking gel buffer (pH. 6.8)
Tris HCl (0.25 M)
3.63 g
SDS (0.2%)
0.2 g
Make the volume to 100 ml with water (MQ)
c. Acrylamide for separating gel
Acrylamide
30 g
BIS
0.135 g
Make the volume to 100 ml with water (MQ)
d. Acrylamide for stacking gel
Acrylamide
29.2g
BIS
0.8g
Make the volume to 100 ml with water (MQ)
e. 1% agarose
1 g of agarose /100 ml of water
f. Running buffer
Tris-HCl
9.0 g
Glycine
43.2 g
SDS
3.0 g
Make the volume to 3 L with water (MQ).
g. Bromophenol blue solution
BPB 0.01 g /100 ml of 10 % glycerol
2.3.2. Method
a. Assembled detergent cleaned gel plates. The separating gel is cast first, followed by
the casting of the upper stacking gel. For a gel with dimension 0.75 mm x 14cm x
14cm, about 20 ml of separating gel mix and about 5ml of stacking gel mix is
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needed.
b. Prepare 15% polyacrylamide gel as shown below
Acryalamide for separating gel
8.5 ml
Separating gel buffer (pH.8.8)
6.3 ml
Water
2.0 ml
10% APS
120 µl
EMED
20 µl
For one plate
Swirl to mix and pour the gel immediately. Gently overlay with 1cm of water. Allow
the gel to polymerize for 45 to 60 minutes
c. Preparation of Stacking gel solution
Stacking gel acrylamide
1.0 ml
Stacking gel buffer
3.0 ml
Water
2.0 ml
APS
30 µl
TEMED
20 µl
Remove the overlaid water and pour the stacking gel solution immediately. Leave it
to polymerize for 10-15 minutes
d. The first dimension is applied directly on the top of the stacking gel. The first
dimension is overlaid by 1% agarose.
e. The slab gel is assembled for running. A few drops of tracking dye (bromophenol
blue) is added
f. Run the sample at 35 mA (constant current) until the tracking dye reaches the bottom
of the separating gel.
g. Separate the gel plates and either stain the gel or process for western blot. The gel is
stained either with silver staining or Coomassie Brilliant Blue
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3. Image Acquisition and Data Analysis
(1) CBB-stained gels were analyzed with Image Master 2D-Elite software. Specially,
spot detection, spot measurement, background subtraction and spot matching were
performed. Following automatic spot detection, gel images were carefully edited.
Prior to performing spot matching between gel images, one gel image was selected as
the reference gel.
(2) After automatic matching, the unmatched spots of the member gels were added to
the reference gel. The amount of a protein spot was expressed as the volume of the
spot which was defined the sum of the intensities of all the pixels that make up the spot.
In order to correct the variability due to CBB staining and reflect the quantitative
variations in intensity of protein spots, the spot volumes were normalized as a
percentage of the total volume in all of the spots present in gel.
(3) The resulting data from image analysis were transferred to 2D Elite software for
querying protein showing quantitative or qualitative variations.
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4. Cleveland Peptide Mapping
(1) Prepared samples are separated by 2D-PAGE. Then the gels are stained with
Coomassie brilliant blue, and gel pieces containing protein spots are removed.
(2) The protein is electroeluted from the gel pieces using an electrophoretic
concentrator run at 2 W constant power for 2 h. After electroelution, the protein
solution is dialyzed against deionized water for 2 d and lyophilized.
a. Cut out stained protein spots from 2D gels and soak in deionized water.
b. Fill 750 μL of electroelution buffer in the 2-mL Eppendorf tube containing protein
spots (5–20 gel pieces). Shake for 30 min.
c. Cut 12- to 15-cm-long seamless cellulose tubing (small size, no. 24, Wako, Osaka,
Japan) as space is needed for clipping. Fill a 300-mL beaker with250 mL deionized
water, boil it for 5 min, and keep the tubing membrane in it. Wet the small pieces of
cellophane film in a small beaker with deionized water.
d. Close the bottom of the small part of cup with a cellophane film, open the bottom of
the large part of cup, connect and twist the tubing membrane, and close the distal
end by clipping (Fig. 1A).
e. Fix the cup in the electrophoretic concentrator (Fig. 1B; Nippon Eido, Tokyo, Japan).
Deposit gel pieces containing proteins on the cellophane film (thesmall part of the
cup), and add 750 μL of the electroelution buffer from the Eppendorf tube. Fill the
small part of the cup with electroelution buffer, and then fill the large part of the cup
with electroelution buffer in such a way thata layer of buffer joins both the cup parts,
allowing movement of protein fromthe small part of the cup to the tubing membrane.
Fill the apparatus with electroelution buffer. The small part of cup containing
protein spots should be toward the positive side.
f. Run at 2 W constant power for 2 h.
g. Remove the tubing membrane and clip to close the end. Dialyze in a cold room
(4°C). Change the deionized water three times the first day. The next day, change
the deionized water two times.
h. Transfer the protein solution to two to six 2-mL Eppendorf tubes. Freeze-dry
overnight.
i. Dissolve the protein in 30 μL of SDS sample buffer.
(3) The protein is dissolved in 20 μL of SDS sample buffer (pH 6.8) and applied to a
sample well of an SDS-PAGE gel. The sample solution is overlaid with 20 μL of a
solution containing 10 μL of Staphylococcus aureus V8 protease (Pierce, Rockford,
IL, USA) (0.1 μg/μL) in deionized water and 10 μL of SDS sample buffer (pH 6.8).
Electrophoresis is performed until the sample and protease are stackedin the
stacking gel. The power is switched off for 30 min to allow digestion of theprotein,
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and then electrophoresis is continued.
a. Fix glasses (100 x 140 x  1 mm) with clip, keeping a 1-mm space between the plates.
b. Prepare separating gel solution in a 100-mL beaker. Mix the solutions, and fill the
plates about 3 cm from top. (Caution: Pour the solutions into the plates immediately
after adding 10% APS and TEMED.)
c. Overlay the separating gel solution with 1 mL water (MQ).
d. Leave the gel for 40 to 60 min at room temperature for polymerization.
e. Remove the overlaid water and pour the following stacking gel solution.
f. Prepare the stacking gel solution in a 100-mL beaker. Mix well, pour on the
separating gel, and insert comb.
g. Leave the gel for 20 min at room temperature for polymerization.
h. Take out the comb, clips, and silicon tubes.
i. Clean the wells with a syringe.
j. Fix the gel plates with the apparatus. Pour SDS-PAGE running buffer.
k. Dissolve the protein in 20 μL of SDS sample buffer (pH 6.8), and apply to asample
well in SDS-PAGE. Overlay the sample with 20 μL of a solution containing10 μL of
Staphylococcus aureus V8 protease with 1 μg/μL in deionized water and 10 μL of
SDS sample buffer (pH 6.8). Add 30 μL BPB solution.
l. Electrophoresis was performed until the sample and protease were stacked in the
upper gel and interrupted for 30 min to digest the protein.
m. Run the gel at 35 mA until the BPB line reaches about 5 mm near the bottom.
n. Disconnect the electricity, and take out the plates.
o. Separate two plates with a spatula.
p. Separate the stacking gel, and take out the separating gel.
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5. Western Blotting
Following separation by 2D-PAGE or by the Cleveland method, the proteins are
electroblotted onto a PVDF membrane (Pall, Port Washington, NY) (Fig. 2), using a
semidry transfer blotter (Nippon-Eido, Tokyo Japan), and detected by CBB staining.
a. Cut the PVDF membrane equal to the size of the gel.
b. Cut the Whatman 3MM filter paper equal to the size of the gel.
c. Wash the PVDF membrane in methanol for a few seconds, and transfer the
membrane to 100 mL blotting solution C; shake for 5 min.
d. Wet two blotting papers (3 MM) in A, B, and C blotting solutions.
e. Place the separating gel in 100 mL blotting solution C, and shake for 5 min.
f. Wet the semidry blotting apparatus with water (MQ). Place blotting paper A on the
blotting plate and then blotting paper B. Remove air bubbles, if any. Place the
PVDF membrane on the plate and then the gel and blotting paper C.
g. Connect the power supply. Run the blotting at 1 mA/cm2 for 90 min.
h. Wash the membrane in 100 mL water (MQ).
i. Stain the gel for 2 to 3 min in CBB stain.
j. Destain the gel in 60% methanol for 3 min twice.
k. Wash with MQ and air-dry at room temperature.
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6. Deblocking of Blotted Proteins
6.1. Acetylserine and Acetylthreonine
Proteins with acetylserine and acetylthreonine at their N-termini separated by
2D-PAGE are electroblotted onto a PVDF membrane. The region of the PVDF
membrane carrying the protein spot is excised and treated with trifluoroacetic acid
(TFA) at 60°C for 30 min. They are then directly subjected to protein sequencing.
6.2. Formyl Group
N-formylated protein separated by 2D-PAGE is electroblotted onto a PVDF
membrane. The region of the PVDF membrane carrying the protein spot is excised and
treated with 300 μL of 0.6 M HCl at 25°C for 24 h. The membrane is washed with
deionized water, dried, and applied to the protein sequencer.
6.3. Pyroglutamic Acid
(1) Proteins with pyroglutamic acid at their N-termini separated by 2D-PAGE are
electroblotted onto a PVDF membrane.
(2) The region of the PVDF membrane carrying the protein spot is excised and treated
with 200 μL of 0.5% (w/v) polyvinylpyrrolidone (PVP)-40 in 100 mM acetic acid
37°C for 30 min (see Note 3).
(3) The PVDF membrane is washed at least 10 times with 1 mL of deionized water.
(4) The PVDF membrane is soaked in 100 μL of 0.1 M phosphate buffer (pH 8.0)
containing 5 mM dithiothreitol and 10 mM EDTA acid.
(5) Pyroglutamyl peptidase (5 μg) is added, and the reaction solution is incubated at
30°C for 24 h.
(6) The PVDF membrane is washed with deionized water, dried, and applied to the
protein sequencer.
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7. N-Terminal and Internal Amino Acid Sequence Analysis and Homology Search
of Amino Acid Sequence
(1) The stained protein spots or bands are excised from the PVDF membrane and
applied to the upper glass block of the reaction chamber in a gas-phase protein
sequencer, Procise 494 or cLC (Applied Biosystems, Foster City, CA, USA), and/or
PPSQ (Shimazu, Kyoto, Japan). Edman degradation is performed according to the
standard program supplied by Applied Biosystems and/or Shimazu. The released
phenylthiohydantoin (PTH) amino acids are separated by an online high-performance
liquid chromatography (HPLC) system and identified by retention time.
(2) The amino acid sequences obtained are compared with those of known proteins in
the Swiss-Prot, PIR, Genpept, and PDB databases with the web-accessible search
program FastA.
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8. Mass Spectrometry
8.1. Preparation of chemicals
Chemicals
Methanol (MeOH) for blotting sequence type
Acetonitrile CH 3 CN), HPLC grade
Trifluroacetic acid (TFA) HPLC grade
Stock solutions
(Preserve at 4 o C)
100 mM NH4 HCO 3
500 mM EDTA.2Na
Washing solution (25% MeOH / 7% Acetic acid)
10 mM Tris-HCl (pH 8.0)
3% TFA
0.1% TFA / 100% CH3 CN
0.1% TFA / 100% H2 O
(Freezer at -20o C)
1 M Dithiothreitol (DTT)
10 pM Trypsin / 10 mM Tris-HCl
Chemicals to be prepared during use
Destaining solution (50 mM NH4 HCO 3 / 50% MeOH)
100 mM NH4 HCO 3 2.5 ml
100% MeOH
2.5 ml
a. Reduction solution ( 10 mM EDTA.2Na / 10 mM DDT / 100 mM NH4 HCO 3
500 mM EDTA.2Na
20 µl
1 M DDT
10 µl
970 µl
100 mM NH4 HCO 3
b. Alkylation solution (10 mM EDTA. 2Na / 40 mM Iodoacetamide / 100 mM
NH 4 HCO 3
500 mM EDTA.2Na
20 µl
Iodoacetamide
7.4 mg
100 mM NH 4 HCO 3
980 µl
c. Trypsin solution
10 pM Trypsin / 10 mM Tris-HCl
50 µl
10 mM Tris-HCl
(pH 8.0)
450 µl
8.2. Methods
a. Cut the gel (spot)
Cut the selected protein spots from the 2D-gels. Place the gels on light box and cut
the protein spots with a spatula. Collect the gel pieces in an eppendorf tube. Wash
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the gel pieces with water (MQ), drain out water. This can be preserved in freezer at
–20 o C.
b. Washing and destaining
Add 1 ml wash solution (25% MeOH / 7% Acetic acid) to the eppendorf tube, fix
them in a rotor and rotate over night at room temperature.
c. Destaining procedure depends on the method of staining:
In case of CBB staining: Wash with water (MQ), add 0.5 ml destaining solution (50
mM NH 4 HCO 3 / 50% MeOH), incubate at 40 o C until the samples (gel pieces) are
destained (may take 30 to 60 min). After destaining, dry out destaining solution then
wash with water (MQ).
In case of silver staining: Add 0.3 ml solution (20 mM EDTA. 2Na / 50 mM
Tris-Hcl (pH8.0) / 30% CH 3 CN), wait for 5 min at room temperature. Drain out
supernatant, repeat this step once more. Then wash with water (MQ) for two times.
d. Reduction and Alkylation
i) Dry the samples in speed Vac. concentrator (may skip this step)
ii) Add 50 µl reduction solution (10 mM EDTA.2Na / 10 mM DTT / 100 mM
NH 4 HCO 3 ) to the eppendorf tube to make the gels wet. Incubate for 1 h at 60 o C
placing on a cool block.
iii) Discard the solution and dry again using speed Vac.
iv) Add 50 l alkylation solution (10 mM EDTA.2Na / 40 mM Iodoacetamide / 100
mM NH 4 HCO 3 ) to the gels in eppendorf tube, place in dark at room temterature for
30 min to carry out reaction.
v) Add 1 ml water (MQ), wash the gels and drain the water. Repeat this step for 2
times.
vi) Smear the gels with a Pelletier.
vii) Dry the gels in a speed Vac. concentrator.
e. Digestion
i) Prepare 1 pmol trypsin solution (10 pM Trypsin / 10 mM Tris-HCl), add 50 µl to
the gels in eppendorf tube.
ii) Wrap the eppendorf tube with Para film, digest at 37 o C for 10 to 12 h.
f. Elution
i) Add 100 µl solution (0.1% TFA / 100% CH 3 CN) to the gels in eppendorf tube(A),
sonicate for 10 min.
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ii) Collect the supernatant in a new eppendorf tube (A1).
iii) Add 100 µl solution (0.1% TFA / 100% MQ) to the gels in eppendorf tube (A),
sonicate for 10 min.
iv) Collect the supernatant in eppendorf tube (A1).
v) Add 80 µl solution (0.1% TFA / 50% CH3 CN) in the gels in eppendorf tube (A),
sonicate for 10 min.
vi) Collect the supernatant in eppendorf tube (A1).
vii) Add 100 100 µl solution (0.1% TFA / 100% CH3 CN) to the gels in eppendorf
tube (A), sonicate for 10 min.
viii) Collect the supernatant in a new eppendorf tube (A1).
ix) Concentrate the collected sample to 20 - 30 µl in a speed Vac. (CH 3 CN will be
evaporated). This sample is peptide solution.
g. Desalt
i) Using reverse phase column, desalt and concentrate the peptide solution.
ii) Wash with ZipTip C18 (Millipore) filling and unfilling 10 µl 0.1% TFA / 50%
CH 3 CN 3 times
iii) Wash and stabilize ZipTip with 10 µl 0.1% TFA / 100% MQ, filling and unfilling
3 times
iv) Take the whole peptide solution in ZipTip (10 µl each time) in repeated times,
thus absorbing the whole peptides in ZipTip columniv) Wash and desalt the ZipTip
with 10 µl 0.1% TFA / 100% MQ, repeat 3 times
v) Dissolve the peptides absorved in column by 0.1% TFA / 50% CH 3 CN. The
sample volume depends on the following operation.
In case of analyzing using VOYAGER-RP:
Use 2 µl peptide on the sample plate and 2 µl Matrix solution, mix them well, air dry
and analyze.
In case of analyzing using other Mass spectrometer
The sample volume depends on requirement.
In case of preservation for future analysis
Take 5 in an eppendorf tube and keep in freezer at -20o C.
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