SDS-Polyacrylamide Gel Electrophoresis
This protocol describes SDS-Polyacrylamide Gel Electrophoresis using the Mini-Protean Gel System (Biorad).
1. Gel Composition
Component
Stacking 4%
7.5%
10%
12.5%
15%
3 600
3 590
2 755
1 925
1 090
1 M Tris.HCl pH 6.8
625
-
-
-
-
1 M Tris.HCl pH 8.8
-
3 750
3 750
3 750
3 750
665
2 500
3 335
4 165
5 000
10% SDS
50
100
100
100
100
10% APS
50
50
50
50
50
TEMED
10
10
10
10
10
5 000
10 000
10 000
10 000
10 000
H 2O
30% Acrylamide
Total
5/10ml is sufficient for two 0.75mm Minigels
2. Gel Preparation and Electrophoresis
• Wipe the glass plates with an ethanol-soaked paper towel. Assemble the glass plate sandwiches as outlined in the
Mini-Protean Instructions and fix them on the casting stand.
• Mix the gel solutions except for APS and TEMED in 15ml Falcon tubes (10ml separation gel and 5ml stacking gel for
two 0.75mm gels).
• Add APS and TEMED to the separation gel solution, mix carefully and pour the gels. This is best done with a pipet
(3.6/4.0ml per gel). Carefully overlay the gel surface with H2O. The gel should polymerize for at least 30min.
• Pour off the overlay and rinse the gel surface with H2O. Dry area above the separation gel by inserting a strip of filter
paper. Insert the comb between the glass plates at a slight angle.
• Add APS and TEMED to the stacking gel solution, mix carefully and pour the gel solution down the spacer nearest
the upturned side of the comb. This is best done with a pipet (0.6ml per gel). Properly align the comb and add
enough gel solution to cover the comb completely. The gel should polymerize at least 30min.
• Remove the comb by pulling it up straight. Remove the gel cassette from the casting frame and assemble it with the
electrode assembly. Make sure that the inner glass plate makes good contact with the U-shaped gasket.
• Position the electrode assembly with the gels so you can clearly see the wells. Fill the upper buffer chamber with 1 x
Running buffer until the buffer reaches a level halfway between the short and the long plates. Check for leaks.
• Sample preparation: Samples in any aqueous buffer should not contain more than 100mM salt. The samples should
contain sufficient protein to be detected by one of the staining methods listed below. If the salt concentration of the
sample is too high or protein concentration is too low, desalting or concentration is required. A simple method that
can be applied to most proteins is TCA precipitation: Add 1/10 volume 100% TCA (trichloroacetic acid) to the
sample. Incubate on ice for at least 1h. Spin 5min 4°C 10000xg. Wash pellet with ice-cold acetone. Spin 5min 4°C
10000xg. Dissolve pellet in a suitable buffer. Add 1/2 volume of SDS-Sample buffer to the sample (50µl per 100µl
sample). Heat 5min at 95°C. Cool to RT. Spin 1min at 10000xg and transfer supernatant to a fresh tube.
• Load the samples by underlayering them with a pipette using gel loading tips. Do not overfill the wells.
• Carefully place the electrode assembly into the lower buffer tank and add 1 x Running Buffer up to the mark.
• Run the gels 5min at 50V and then 60-75min at 150V until the bromphenol blue reaches the bottom of the gel.
• Remove the electrode assembly from the lower buffer tank and disassemble the gel assembly. The glass plates are best
separated using the plate separation tool from Biorad. Cut away the stacking gel and mark the lower right corner of
the separation gel. Place the gel in a tray for staining or equilibration in blotting buffer.
3. Gel Staining
a. PageBlue Stain or Colloidal Coomassie Stain
• Fix gel 30min in 10% TCA with slight agitation.
• Wash gel 10min with H2O by heating for 20s in microwave and subsequent slight agitation.
• Stain gel 1-16h with PageBlue or Colloidal Coomassie with slight agitation.
• Destain gel 3-4 x 10min with H2O by heating for 20s in microwave and subsequent slight agitation until the
background becomes clear.
1
b. Hot Silver Stain
• Fix gel 0.5-16h (longer fixation reduces background) in 10% TCA at 4°C with slight agitation.
• Soak gel 5min in H2O, drain.
• Add 50ml H2O, microwave for 20s, incubate at RT with slight agitation for 5min, drain.
• Add 50ml 0.2mM DTT (20µl 1M DTT/100ml), microwave for 20s, incubate at RT with slight agitation for 5min,
drain.
• Rinse gel two times for 10s with 100ml H2O.
• Add 50ml 0.1% AgNO3 (0.1g/100ml), microwave for 20s, incubate at RT with slight agitation for 5min, drain.
• Rinse gel two times for 10s with 100ml H2O.
• Add 20ml Developer, shake well until a brownish precipitate begins to form, then pour off; add 20ml fresh Developer,
shake for 30s, then pour off; add 60ml fresh Developer, develop with slight agitation until the signal to background
ratio is optimal (usually 3-5min), then pour off Developer.
• Add 30ml 20% Citric acid, incubate at RT with slight agitation for 10min.
• Soak gel in H2O with slight agitation for 10min and then dry the gel.
c. Cold Silver Stain
• Fix gel 0.5-16h (longer fixation reduces background) in 10% TCA at 4°C with slight agitation.
• Soak gel 5min in H2O, drain.
• Add 50 ml H2O, incubate at RT with slight agitation for 20min, drain.
• Add 50ml 0.2mM DTT (20µl 1M DTT/100ml), incubate at RT with slight agitation for 20min, drain.
• Rinse gel two times for 10s with 100ml H2O.
• Add 50ml 0.1% AgNO3 (0.1g/100ml), incubate at RT with slight agitation for 20min, drain.
• Rinse gel two times for 10s with 100ml H2O.
• Add 20ml Developer, shake well until a brownish precipitate begins to form, then pour off; add 20ml fresh Developer,
shake for 30s, then pour off; add 60ml fresh Developer, develop with slight agitation until the signal to background
ratio is optimal (usually 3-5min), then pour off Developer.
• Add 30ml 20% Citric acid, incubate at RT with slight agitation for 10min.
• Soak gel in H2O with slight agitation for 10min and then dry the gel.
4. Gel Drying
• Gels should be soaked in H2O for at least 10 min prior to drying.
• Put one sheet of dry blotting type filter paper (35x45cm) on the porous gel support of the gel dryer.
• Drying onto Filter Paper: Make up a sandwich consisting of a sheet of wet filter paper (75x110mm), the gel, and a
cover of nonporous plastic film (such as Saran Wrap) directly on the dry filter paper. Avoid to trap any air bubbles
between the wet filter paper, the gel and the cover.
• Drying between sheets of Cellophane Membrane: Pre-wet the Cellophane membrane (75x110mm) for 5min in H2O.
Make up a sandwich consisting of a piece of cellophane, the gel, a second piece of cellophane, and a cover of
nonporous plastic film (such as Saran Wrap) directly on the dry filter paper. Avoid to trap any air bubbles between
the cellophane, the gel and the cover.
• Lay the transparent sealing gasket over the sandwich and close the lid of the gel dryer. Turn on the vacuum.
• Select the appropriate cycle, temperature, and time (see below) and start drying.
• When the timer turns off the heating element, open the lid to cool the gel. When the gasket is cool to touch, open the
gasket and remove the gel.
• Drying time is a function of temperature, vacuum, gel porosity, gel thickness and number of gels. Using the Bio-Rad
Model 583 Gel Dryer with theVacuubrand MZ2C vacuum pump use the following conditions for drying 4 standard
0.75 mm gels ( increase time for more gels accordingly):
Gel
Medium
Cycle
Temperature
Time
< 15%
Filter Paper
normal
80°C
45 min
< 15%
Cellophane
normal
80°C
90 min
≥ 15%
Filter Paper
slow
60°C
90 min
≥ 15%
Cellophane
slow
60°C
120 min
5. Reagents
• 1M Tris.HCl pH 6.8:
Tris(hydroxymethyl)aminomethan
H 2O
pH=6.8 (HCl)
filter (0.45µm), store at 4°C
2
12.11g
to 100ml
• 1M Tris.HCl pH 8.8:
Tris(hydroxymethyl)aminomethan
H 2O
pH=8.8 (HCl)
filter (0.45µm), store at 4°C
60.55g
to 500ml
• 30% Acrylamide:
Acrylamide
29.19g
N,N´-Methylene-bis-acrylamide
0.81g
to 100ml
H 2O
filter (0.45µm), store dark at 4°C (max. 3 months)
• 10% SDS:
Sodium dodecylsulfate
H 2O
filter (0.45µm), store at RT
• 10% APS:
Ammoniumpersulfate
H 2O
store at 4°C (max. 1 week) or at -20°C
• TEMED:
N,N,N´,N´-Tetramethyethylenediamine
store dark at 4°C
• SDS-Sample Buffer:
60mM Tris.HCl pH 6.8
6ml M Tris.HCl pH 6.8
6% SDS
6g
6mM EDTA
1.2ml 0.5M EDTA pH 8.0
10% Glycerol
10g
0.05% Bromphenol blue
50mg
H 2O
to 95ml
filter (0.45µm), store at RT
5% 2-Mercaptoethanol (to be added immediately before use)
This buffer is 3 x concentrated; add 50µl per 100µl of sample.
• 10 x Running Buffer:
Tris(hydroxymethyl)aminomethan
Glycine
SDS
H 2O
pH 8.3 (unadjusted), store at RT
30g
144g
10g
to 1000ml
• 1 x Running Buffer:
Tris(hydroxymethyl)aminomethan
Glycine
SDS
H 2O
pH 8.3 (unadjusted), store at RT
3g
14.4g
1g
to 1000ml
• 10% TCA:
Trichloroacetic acid
H 2O
100g
to 1000ml
• PageBlue:
PageBlue Protein Staining Solution (Fermentas)
• Colloidal Coomassie:
Ammonium sulfate
100g
Phosphoric acid 85%
23.5g
to 880ml
H 2O
Add 1g Coomassie brilliant blue G-250 dissolved in 20ml H2O to the solution while stirring.
Store dark at RT, add 10% Methanol immadiately before use.
This is a homemade version of the PageBlue Protein Staining Solution.
• 0.2mM DTT:
1M DTT
H 2O
• 0.1% AgNO3:
1% AgNO3
H 2O
• Developer:
Na2CO3 (30g/l)
37% Formaldehyde
• 20% Citric acid:
Citric acid
H 2O
10g
to 100ml
100mg
1ml
20µl
100ml
10ml
to 100ml
100ml
50µl
200g
to 1000ml
CONTRIBUTED BY: Hubert Schwelberger (hubert.schwelberger@i-med.ac.at)
LAST MODIFIED: 2012-06-21
3