6.@ Western blotting

Yuji Hiwatashi

 

Introduction

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

Procedure

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).

 

Procedure

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.

 

Solution required

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

Procedure

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.

 

Solution required

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)