This protocol
describes (1) protein extraction from P. patens, (2) SDS-PAGE, (3)
blotting and (4) immunodetection of membrane-blotted proteins.
(1) Protein
extraction
Two ways: a)
extraction with SDS-sample buffer (the simplest method) and b) extraction from
TCA precipitate for relative high molecular protein such as kinesin and myosin.
a)
Extraction with SDS-sample buffer
1. Add 500
µl of 2X SDS sample buffer to a 15 ml conical tube.@
2. Grind tissue
(for example, protonema < 100 mg fresh weight) in liquid nitrogen using
mortar and pestle.@ Transfer the fine
power of tissue to the 15 ml conical tube.@
Vortex.
3. Transfer the
mixture to a 1.5 ml micro tube.
4. Heat at
100˚C for 3 min.
5. Centrifuge at
15000 rpm for 10 min.
6. Recover the
supernatant as a crude protein fraction.
b)
Extraction with TCA
This protocol
is used for immunodetection of myosin and kinesin (Yokota
and Shimmen (1994) Protoplasma 177: 153-162).
1.
Grind Tissue (~ 50 mg tissue, DO NOT excess ~0.1 g) in 0.5 ml of TCA
solution on ice.
2.
Transfer the homogenate into a micro tube
3.
Centrifuge at 10000 rpm for 5 min at 4˚C and remove the
supernatant.
4.
Suspend the pellet in 1 ml acetone/Tris (pipetting and vortex).
5.
Centrifuge at 10000 rpm for 5 min at 4˚C and remove the
supernatant.
6.
Suspend the pellet in 1 ml acetone/Tris (pipetting and vortex).
7.
Centrifuge at 10000 rpm for 5 min at 4˚C and remove the
supernatant.
8.
Suspend the pellet in 0.5 ml acetone/Tris (pipetting and vortex).
9.
Centrifuge at 10000 rpm for 5 min at 4˚C and remove the
supernatant.
10.
Soak the pellet in 1 ml of 100 mM Tris HCl pH 6.8 for 5 min on
ice.
11.
Centrifuge at 10000 rpm for 5 min at 4˚C and remove the
supernatant.
12.
Suspend the pellet in 0.5 ml 1x SDS-PAGE sample buffer (+5%
2-mercaptoethanol) and keep on ice for 15 min.
13.
Heat the sample solution for 5 min at 105˚C.
14.
Centrifuge at 13000 rpm for 5 min at room temp.
15. Recover
sup and go to SDS-PAGE step.
1.@@@ 15% (w/v) TCA
solution
stock
reagent |
/
100 ml |
final
concentration |
Trichloroacetic
acid |
15
g |
15% |
H2O |
Fill
up to 100 ml |
|
stored
at 4C@
2.@@@ Acetone/Tris
stock
reagent |
/
100 ml |
final
concentration |
Acetone |
80
ml |
80%
(v/v) |
1
M Tris HCl pH8.0 |
5
ml |
50
mM |
H2O |
Fill
up to 100 ml |
|
stored
at 4˚C@
3.@@ 100 mM Tris HCl pH 6.8
4.@@@ 2x SDS-PAGE sample
buffer (-ME)
stock
reagent |
/
9 ml |
final
concentration |
1
M Tris-HCl (pH6.8) |
1.25
ml |
125
mM |
10%
SDS |
4
ml |
4% |
BPB |
2
mg |
0.02% |
Glycerol |
2
mg |
20% |
H2O |
Fill
up to 9 ml |
|
7. 2-mercaptoethanol
(2) SDS-PAGE
This
protocol describes preparation of a mini-gel (10 x 10 cm)
1. Clean
glass plates with ethanol and assemble casting stand.
2. Mix
each reagent (see table below) in 50 ml beaker.@ Mix well.
|
10% gel |
10% gel x 2 |
7.5% gel |
7.5% gel x
2 |
30%
acrylsmide/Bis |
2.35 ml |
4.7 ml |
1.75 ml |
3.5 ml |
4x lower
buffer pH8.8 |
1.75 ml |
3.5 ml |
1.75 ml |
3.5 ml |
H2O |
2.84 ml |
5.68 ml |
3.44 ml |
6.88 ml |
10 % APS |
70 µl |
140
µl |
70 µl |
140
µl |
TEMED |
7 µl |
14 µl |
7 µl |
14 µl |
3. Add
7 µl of TEMED and mix well.
4. Pour
the solution between the plates until 2cm from the top end. @There should be a sufficient room for upper
gel and comb.
5. Carefully
overlay 0.1% SDS and allow to set. @Once
the gel has set it can be wrapped in wrap and stored for several days at 4˚C.
6. Mix
the following solutions for upper gel (see table below) in 50 ml barker or
flask and mix well.
@375 µl 30%
acrylamide/Bis@@@@@@@@@@@@@@@@
@562.5 µl 4 x upper
buffer (pH6.8)@@@@@
@1290 µl H2O@@@@@@@@@@@@@@@@
@22.5 µl 10% AMPS
7. Add
4.5 µl of TEMED into the solution and mix well.
8. Overlay
upper gel immediately. Insert comb and allow to set for 15-20 min.@ The upper gel should not be prepared until
the samples are ready as there is a pH difference between the two gels which
will diffuse with time.
9. Add
100 µ of 2x sample buffer supplemented with 2-mercaptoethanol (final
10%)into 100 µl protein sample if you mix the sample with the sample
buffer.@ Vortex well and spin down.@
10.
Heat at 100˚C for 3 min and spin down.@ Dilute 40 ml of 10X SDS running buffer to
360 ml of H2O while the samples are denatured.
11.
Load 5-20 µl of the sample.@ Load 10 µl sample buffer to residual
lanes to prevent smiling. @Flood the
upper chamber by carefully adding 1 x SDS running buffer. @Avoid pouring the buffer directly onto the
wells.
12.
Run a gel at 10-15 mA until dye front goes to
the bottom of stacking gel and then run at 30 mA.
13.
Wash gel with H2O for 5 min x 3
times.@ Then, stain for 60 min with Bio-safe
Coomassie BioRad (cat.no.161-0786). After staining, wash the gel with H2O
for more than 1 h.@@
Instrument
required
E Electrophoresis
apparatus
E Gel plate
and gasket
Solution
required
1.@@@ 4x Tris/SDS (pH 6.8)@ For stacking gel
stock
reagent |
/ 100 ml |
final
concentration |
Tris |
6.05 g |
0.5 M |
SDS |
0.4 g |
0.4% |
Adjust to
pH6.8 with HCl |
|
|
H2O |
Fill up to
100 ml |
|
Filtrate with
0.45 um filter and store at 4˚C@
2.@@@ 4x Tris/SDS (pH 8.8)@ For running gel
stock
reagent |
/ 250 ml |
final
concentration |
Tris |
45.5 g |
1.5 M |
SDS |
1 g |
0.4% |
Adjust to
pH8.8 with HCl |
|
|
H2O |
Fill up to
250 ml |
|
Filtrate with
0.45 um filter and store at 4˚C@
3.@@ 10% SDS
4.@@@ 10 x Reservoir buffer
stock
reagent |
/ 500 ml |
final
concentration |
Tris |
15.15 g |
0.25 M |
Glycine |
72.5 g |
1.9 M |
SDS |
5.0 g |
1% |
H2O |
Fill up to
500 ml |
|
5.@@@ 2x SDS-PAGE sample buffer (-ME)
stock
reagent |
/ 9 ml |
final
concentration |
1 M
Tris-HCl (pH6.8) |
1.25 ml |
125 mM |
10% SDS |
4 ml |
4% |
BPB |
2 mg |
0.02% |
Glycerol |
2 ml |
20% |
H2O |
Fill up to
9 ml |
|
6.
30% acrylamide-0.8% bis-acrylamide
Bio-Rad@@ 30% acrylamide/Bis solution, 37.5:1@ (Cat.no.161-0518)
7.
TEMED (amresco: ultra pure grade)
8.
10% APS
9.
Bio-safe Coomassie BioRad (cat.no.161-0786).
(3) Blotting
Procedure
1.
Incubate the gel after electrophoresis in the blotting
buffer.@ During incubation, soak the
PVDF membrane in methanol for a few minutes. Remove methanol and add 1x
Blotting buffer until ready to use.
2.
.Pre-wet the sponges, filter papers (slightly bigger than gel) in
1x Blotting buffer. Assemble in the following order: blotter – sponge -
filter paper - gel - membrane - filter paper – sponge.
3.
Transfer for 3 hr at 80 V or overnight at 16 V in the cold room by
a wet type blotting apparatus.
4. Immerse
the blotted membrane in blocking buffer for 1 h at room temp or overnight at
4˚C.
Instrument
required
E Blotting
apparatus (wet type)
E Sandwich
box
E Chromatography
paper
Solution
required
E Blotting
buffer (25 mM Tris, 192 mM glycine, 10% (v/v) methanol)
E Methanol
E PVDF
membrane (Hybond P: Amersham)
(4) Immunodetection
1. After
blocking, incubate with primary antibody diluted in blocking buffer for 60 min
at room temp.
2. Rinse
3 times with TBS-T and then wash 3 x 10 min with TBS-T.
3. Incubate
with secondary antibody diluted in blocking buffer (ex. Anti-mous IgG-AP
conjugate [NEB, cat.no. 7052-1], we use 1:5000-diluted working solution) for 60
min at room temp.
4. Rinse
3 times with TBS-T and then wash 3 x 10 min with TBS-T
5. Detect
with Vector Alkaline phosphatase substrate kit.
E Skimmilk
E TBS+0.1%
Tween-20 (TBS-T)
E Blocking
reagent (TBS-T supplemented with 0.5% skimmilk)
E Primary
antibody
E Secondary
antibody (Alkaline phosphatase-conjugated, Anti-mous IgG-AP conjugate [NEB,
cat.no. 7052-1])
E BCIP/NBT
Alkaline phosphatase substrate kit IV (Vector laboratory: cat.no. SK-5400)