1. Protein Extraction 

Extraction buffer : (1X SDS PAGE 100mL)

5mL 1.5M Tris-HCl pH 6.8
3mL 20% SDS
15mL Glycerol
Adjust to 100mL, store aliquots at -20°C

+ (optional) : add protease inhibitor cocktail  (6000x Sigma ref P8215) before use.

Inoculate 4mL YPD with one single colony.
Let grow overnight at 30°C with shaking.
Inoculate 1mL of the o/n culture in 50mL YPD
Let grow appromimatly 4h at 30°C with shaking to have OD600=0.5 .
Centrifugation 5 min at 3500 rpm to pull down cells
Cells washed in 500µL extraction buffer. Transfer to 2mL Eppendorf tubes.
Centrifugation at max speed for 1 min.  Remove supernatant (discard)
Cells are now resuspended in 200µL of extraction buffer.
Microbeads are added (same volume as cells)
Vortex 1h à 4°C in order to discrupt cells.
Centrifuge at max speed for 5 min (+4°C) and keep supernatant that contains proteins.
On theVibraCell 75185 (modèle 130W, amplitude 40%) sonicate the supernatant (in water and ice) 3X10sec
Centrifuge at max speed for 1 min (+4°C) and keep supernatant that contains proteins.
Freeze at -80°C

Protein concentration can be measured later. (Bradford method or other). Usual yield is about 20µg.
NB : always work on ice to protect proteins from degradation.

 

To dose your proteins (Bradford)

We use Bio-Rad Protein Assay (#500-0006) 

 

Make a linear standard with BSA (from NEB tube) stock 10mg/mL. Make a dilution at 1µg/µL and keep 50µl aliquots at -20°C. Typical standard of BSA concentration (µg)    : 10, 8, 4, 2, 1, 0

NOTE: SDS interfeers with Bradford, so add 1µL of extraction buffer to every standard tube, including the zero. 

Dose your standards :
800µL H2O+ x µL BSA+1µL extraction buffer +200µl BioRad Protein Assay
Dose your samples:
800µL H2O+1µL sample+200µl BioRad Protein Assay

 

Measure on spec at 595nm. We need a minimum of 10µg for Western Blot Analysis.

 

 

2. SDS-PAGE Gel Preparation

Wash the glass plates first with water and soap, then with ethanol. Place and secure them on the casting frame. 

 

The composition of gels is indicated on this pdf sheet . Adapt gel concentration to the desired size-resolution (e.g. 15% is fine for histones).

 

RUNNING GEL: to be prepared first.

 

Prepare over 5mL per  gel. Immediately after mixing the polymerizing agents, pour between the plates up to the marks. Keep in mind that the gel will retract a little bit once solidified. Add water atop and gently tap the system to help the gel settling down neatly. Your gel is ready when the leftovers in the falcon tube are. (takes about 30min).

 

STACKING GEL:

 

Remove water and dry with absorbing paper. Perpare 1mL of stacking gel, pour it atop the running and immediately add the comb. Let solidify.

 

 

3. Electrophoresis

Loading mix (5x) (store at -20°C):

5.83 mL Tris pH 6.8
3.64 mL Glycérol 87%
0.84g SDS
0.78g DTT
3 mg bromophenol blue
add H2O to 10mL.

Protein denaturation: 2.5µL Loading mix+ 12µL sample (in extraction buffer) : 5 min at 95°C (or 10 min at 80°C)

 

Migration buffer:

100mL Laemmli 10X (MCM)
 2mL SDS 20%
 H2O to 1L

If you're running and odd number of gels, add a clean mock plastic plate.

Place the gels inside the chambers, making sure the wells' side face the interior of the chamber. Fill the compartments with migration buffer, above the wells and above the marks.

Load samples in wells using 'elongated' pipet tips or a Hamilton seringe.

Don't forget the ladder size marker (e.g. Marker page ruler prestrain protein ladder): 5µL.

Run the gel at constant voltage 100V for about 2hours, keeping an eye on the migration line.

 

4. Gel Transfer

Prepare Whatman papers and membrane with same size as gel.

Your choice of membrane can be: 

*PVDF (higher sensitivity) works with ECL or ECL+
*Nitrocellulose (lower sensitivity) works with ECL only. 

0.45µM membranes should be fine for a wide range of protein sizes. Although we were recommended to use 0.2µM membranes for small proteins, we observed histones on 0.45µM nitrocellulose membranes (code M26 in stock)just as fine.

 

Transfer Buffer:

100mL Laemmli 10X (MCM)
200mL Ethanol technique 100%
5mL SDS 20%

H2O to 1L.

Prepare a bath of transfer buffer to immerge membrane, papers, sponges, and gel BEFORE use.

 

Make a sandwich in the following order starting from the black side:
- one sponge

- three papers

- gel

- membrane 

- three papers. Remove possible bubles by rolling a pipette atop the papers.

- sponge 

Close the sandwich.

 

Load in chamber. Transfer is optimal if high molecular weight are at bottom. Add a magnetic barrel. Run at 4°C or add an ice block and run at room temperature. Transfer at 100V for 1 hour for small proteins or 80V for 2 hours for big ones. 

If you need/want to verify transfer efficiency:

- First check that the ladder transfered correctly.

 

- You can then use Ponceau labelling (15g TCA + 1g Ponceau Red + water to 500 mL, recycle if you want).

Incubate membrane 15min at Room Temp. 

Wash membrane with water at Room Temp. for 30 min, changing water regularly.

 

- Alternatively, you can stain the gel with coomasiee-blue to estimate what's left in it:

Staining solution : 250mg Coomassie Brillant Blue + 100mL EtOH/acetic acid solution

EtOH/acetic acid solution : 500mL EtOH 100%+ 400mL H2O+ 100mL acetic acid (!!! FUME HOOD !!!)

Stain for 1h at room temp. Recycle the solution if you want.

Wash with repeated bathing in EtOH/acetic acid solution for between 4 hours to overnight.

5. Blocking and immuno-staining

 

 Wash membrane in  TBS+Tween 0.02% during 10-15 min at room temp.

Transfer it to 5% milk in TBS and incubate for 1 hour at room temp.

Rince  3X 10 min en TBS + Tween 0.02

Incubate with primary antibody  in TBS +  0.3% milk overnight at 4°C (10mL). See recommendations coming with your antibody for concentrations and particularities.

Rince  3X 10 min en TBS + Tween 0.02%

Incubate with secondary antibody  in TBS +  0.3% milk for 1 hour at room temp (10mL) 

Rince  3X 10 min en TBS + Tween 0.02%

5. Detection with ECL kit  

 

Mix 1mL of RA with 1mL of RB on a saran plastic wrap.

Dry membrane (no scratch!) shortly and place on the mixture (protein size down). Move it gently to incubate evenly. After 1 min, dry the membrane shortly to remove excess liquid, and place it in cassette, protein side up.

 

In dark room: Try various exposure times:  1 min, 5 min, 10 min

Machine: put films in the proper orientation (entry front = short border, long border against the wall) and press button if this is the first film. Wait for the beep before switching the light on or before placing another film.

Film treatment typically takes about 3 minutes.

 

Write on film: date, exposure time, antibody and ladder. 

Dernière mise à jour : ( 20-01-2009 )