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 a TCA precipitate for relatively high molecular weight proteins
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 a mortar and pestle.@ Transfer the
finely powdered 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 exceed ~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
supernatant 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 |
|
5. 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% acrylamide/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. @There
should be a sufficient room for upper gel and comb.
5.
Carefully overlay with
0.1% SDS and allow to set. @Once the gel
has set it can be wrapped in Saran wrap and stored for several days at 4˚C.
6.
Mix the following solutions
for upper gel (see table below) in 50 ml beaker 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% APS
7.
Add 4.5 µl of
TEMED into the solution and mix well.
8.
Overlay the lower gel
with upper (stacking) gel solution 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 µl of 2x
sample buffer supplemented with 2-mercaptoethanol (final 10%) to 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 with 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 the gel at 10-15
mA until dye front reaches the bottom of the 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 |
|
Filter 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 |
|
Filter 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. After
electrophoresis, incubate the gel 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 using 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 (eg. Anti-mouse 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-mouse IgG-AP conjugate [NEB,
cat.no. 7052-1])
E BCIP/NBT
Alkaline phosphatase substrate kit IV (Vector laboratory: cat.no. SK-5400)