SDS PAGE and WESTERN BLOTTING

SDS PAGE

1 Assemble the glass plates, spacers and combs.

2 Prepare the appropriate volume of solution containing the desired

concentration of acrylamide (see Ref: Antibodies a laboratory
manual, page 639). Mix the components in the order shown and
then swirl the mixture rapidly.

3 Pour the acrylamide solution into the gap between the glass
plates. Leave sufficient space for the stacking gel (the length of
the teeth of the comb plus 0.5 cm).
Using a pipette, overlay the acrylamide solution with distilled
water.

4 After polymerization is complete (circa 30 minutes), pour off the

water.

5 Prepare the stacking gel containing the appropriate amount of

acrylamide, using the volumes giving in table (Ref: Antibodies a
laboratory manual, page 639).

6 Pour the stacking gel mixture onto the surface of the previous
gel, place the appropriate comb into the stacking gel. Leave it to
polymerize (circa 15 minutes).
The gel can be kept at 4oC wrapped in a wet tissue and Saran
wrap.

7 Place the gel into the electrophoresis chamber and fill the
chambers with Laemmli buffer (1x).

8 Remove the combs from the stacking gel, wash the sample wells
with Laemmli buffer by pipetting the buffer up and down.

9 Dissolve the protein sample in equal volume of 2x Laemmli

sample buffer (for very diluted proteins use 4x concentrated
sample buffer).

10 Heat the sample at 95oC for 5 minutes and load the samples into
the bottom of the wells.

11 Connect the chamber to the power supply and apply a voltage of

60 until the samples reach the separating gel. At this time,
increase the voltage to 120.
12 When the dye front reaches the bottom of the gel turn off the
power supply.

13 Remove the glass plates from the electrophoresis chamber and

mark the orientation of the gel by cutting a corner from the gel.
Gels are now ready for fixing or transfer of the protein to a solid
support.

COOMASSIE STAINING

1 Fix and stain the gel with Coomassie staining solution for 4 hours
to overnight in a slowly shaking platform. (for a faster staining,
20 minutes at 50-60oC)

2 To visualize the band destain the gel by successive incubations in

Coomassie destaining solution. (for a faster destaining, 1-2 hours
at 50-60oC)

WESTERN BLOT

1 Cut four pieces of Whatman 3MM paper and one piece of NC or

PVDF membrane to the exact size of the SDS-polyacrylamide gel.

2 Wet the filter papers and the membrane in transfer buffer (in the
case of PVDF soak before in 100% methanol).

3 Place two of the Whatman papers that have been soaked in

transfer buffer onto the anode. Stack the sheets one on top of
the other so that they are exactly aligned. Using a glass pipette
as a roller, squeeze out any air bubbles.

4 Place the membrane on the stack of Whatmann paper. Make sure

that the filter is exactly aligned and that no air bubbles are
trapped between it and the Whatman paper.

5 Transfer the gel on top of the membrane. Squeeze out any

trapped air bubbles.

6 Place the final two sheets of Whatman paper on the gel and cover
the sandwich with the cathode.

7 Semi-dry blot the protein at circa 2mA/cm2 of the membrane for

1 hour, i.e. 150 mA for a small size gel. Alternatively, 250 mA for
30 minutes.

8 Transfer the gel to blocking buffer [PBS with 5% milk]

(the membrane can be kept at 4oC)
 9 Detection with ECL. Amounts are for a small format gel (8x6 cm)
(Attention: Never add Na azide to PBSM when using
HRP!!!!)

Blocking 30 minutes with ~20 ml PBSM

First Antibody binding 2 h with the appropriate Ab dilution in
PBSM
Wash 3x10 minutes with ~20 ml of PBST
Second Antibody binding A) 1h with HRP-conjugated Ab in PBSM
Wash 3x10 minutes with 20 ml of PBST
Substrate reaction 1 minute with ECL developing mixture
Detection Saran wrap and expose to X-ray film
(1-30 minutes)

REFERENCES

E. Harlow and D. Lane (1988). Antibodies: a laboratory manual. Cold

Spring Harbor Laboratory.

Laemmli, E. K. (1975) Nature 227, 680-685.

Solutions for SDS PAGE

30% Acrylamide solution (37.5:1; Acryl:Bis)

Keep at 4°C protected from light.

10% APS (ammonium persulphate) solution.

Keep at -20°C in aliquots. Use only freshly thawed.

TEMED (Tetramethylethylenediamine)

10% SDS solution

*1.5M Tris pH 8.8 [Separating gel buffer]

*1.0M Tris pH 6.8 [Stacking gel buffer]

10x Laemmli running buffer: for 1l:

250mM Tris base 30.3g
1.9M Glycine 144.2g
0.1% SDS 10g
ddH2O to 1l (adjust pH to 8.3)

2x Laemmli sample buffer: for 10ml:

20% Glycerol 2ml
2% SDS 2ml of 10% stock solution
250mM Tris pH 6.8 2.5ml of 1M stock solution
10% ß-Mercaptoethanol 1ml
0.1% Bromophenol Blue a pinch!
2.5 ml ddH2O

Coomassie staining solution:

40% Methanol
10% Acetic acid
50% ddH2O
0.25% Coomassie Brilliant Blue R250
Dissolve Brilliant Blue in methanol before adding acid and water.

Coomassie destaining solution:

40% Methanol
10% Acetic acid
50% ddH2O
Solutions for WESTERN BLOTTING

Semi-dry transfer buffer: for 1l:

48mM Tris 5.8g
39mM Gycine 2.9g
0.0375% SDS 0.37g
20% Methanol 200ml
ddH2O to 1l

*10x PBS: for 1l:

1.37M NaCl 80g
27mM KCl 2g
80mM Na2HPO4 11.5g
18mM KH2PO4 2.4g
pH should be 6.8

Blocking buffer [PBSM]:

5% Milk powder (low fat) in PBS
Prepare fresh each time!

ECL detection reagent number 1 (Amersham)

ECL detection reagent number 2 (Amersham)
ECL developing mixture (1:1) of each ECL reagent just before developing.

All solutions kept at room temperature, unless otherwise indicated.

* Solutions should be autoclaved or prepared from sterile stock solutions.