SDS-PAGE
Content
Introduction .............................................................................................................................................2
Materials for SDS-PAGE .........................................................................................................................3
Method for SDS-PAGE ...........................................................................................................................4
Preparing sample ....................................................................................................................................4
Resolving gel preparation .........................................................................................................................5
Stacking gel preparation ...........................................................................................................................6
Protein loading into gel ............................................................................................................................8
Stain and destain the gel ........................................................................................................................10
1
SDS-PAGE
INTRODUCTION
Polyacrylamide gel electrophoresis (PAGE) is a commonly used technique to separate proteins.
The most widely used method was developed by Laemmli using the denaturing (SDS)
discontinuous method. This protocol relies on the presence of SDS (sodium dodecyl sulfate)
and ß-mercaptoethanol to denature the proteins, dissociate the proteins into subunits, and to coat
them with negative charges (SDS). This relatively uniform charge to mass ratio allows the
proteins to migrate in an electric field and separate according to mass/size. The Laemmli
system uses buffers of different pH and composition to generate a voltage gradient and a
discontinuous pH between the stacking and the resolving gel. A 4% acrylamide stacking gel (pH 6.8)
is poured on top of a 10% acrylamide resolving gel (pH 8.8). The stacking gel (with a large
pore size) serves to concentrate all of the proteins (the large ones can catch up with the small ones) on
top of the resolving gel. After entering the resolving gel (which has a smaller pore size) the proteins
are separated according to relative molecular size.
2
SDS-PAGE
MATERIALS FOR SDS-PAGE
x8.3mm)
-well) (0.75 or 1.5mm)
3
SDS-PAGE
METHOD FOR SDS-PAGE
1. Preparing sample:
The sample to be analyzed is mixed with SDS, ananionic detergent which denatures secondary
and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in
proportion to its mass. Heating the samples to at least 65°C further promotes protein
denaturation, helping SDS to bind
a) Mix protein 4:1 with the sample buffer. Heat your sample by either:
b) Boiling for 5-10 minutes (Works for most proteins)
2. Pouring the resolving gel
a) Clean glass plates with soap and water, then with ethanol. Assemble the glass plates and
spacers.
b) For large gels, seal with 1 mL of resolving gel. Remove 2 mL and when sealant is polymerized,
the remaining 1 mL of gel will be polymerized. Add to 2 mL of resolving gel, 4 mL TEMED
and 60 mL 10% APS to polymerize sealant. Seal the large gel plates by pouring 1 mL of
resolving gel (after adding the APS/TEMED) into the plates and tilting them to spread the
sealant. Allow most of excess sealant to run out. While this is going on, degas (for 10 minutes)
the remaining quantity of unpolymerized resolving gel. For the minigel, attach the glass plate
assembly to the apparatus and place the whole gimisch in a small, flat tupperware container.
4
SDS-PAGE
With apasteur pipette, seal the bottom of the plates by spreading 1% agarose in SDS PAGE
electrophoresis buffer along the bottom of the glass plates so it goes up the crack by capillary
action.
c) Transfer the degased resolving gel to a beaker and add APS/TEMED. Quickly add the
resolving gel solution to the center of the plates to a height about 4 cm from the top for the
large plates. For the minigel, add the resolving gel with a pipette.
d) Quickly add isobutanol to the top of this until the level reaches the top of the plates.
5
SDS-PAGE
Isobutanol will prevent oxygen from getting into the gel which could oxidize it and inhibit
polymerization.
e) The resolving gel should polymerize in 30 minutes.
f) Add the stacking gel reagents to a flask and degas for ten minutes.
3. Pouring the stacking gel
a) Pour off isobutanol. Pour water several times into the gel plate space to rinse off all the
isobutanol.
6
SDS-PAGE
b) To polymerize the stacking gel, add APS/TEMED, mix, then pour on top of the polymerized
resolving gel.
c) Insert the comb straight on down, then pour a little more stacking gel on the sides of
the comb to fully seal it.
Remove any bubbles from underneath the comb, if possible, by moving the comb gently from
side to side so the bubbles get into the space in between and float up.
d) The stacking gel should polymerize in 20 to 30 minutes.
7
SDS-PAGE
4. Load the gel
a) Attach the large gel plates containing the polymerized gel to the apparatus via the clips
provided.
b) Pour Tris-glycine electrophoresis buffer into the upper and lower chambers. The large gel
takes about 1 liter, the minigel takes about 200 mL.
c) Remove bubbles trapped at the bottom of the glass plates in the large gel with a syringe.
d) Flush the wells with a syringe just before loading to get rid of any unpolymerized
polyacrylamide that may seep in.
8
SDS-PAGE
e) When loading the gel, load something (1X loading buffer in blank lanes) in every lane
and the dye front will migrate more evenly.
f) Run large thin gels at a constant current of 20 mA. After the dye front enters the
resolving gel, you can turn the current up to 30 mA. Where you stop the gel will depend on how
big the smallest protein is that you want to visualize. If you wait until the dye front just flows out of
the gel, it should take about 2 1/2 - 3 hours for a large, thin gel to run. A thick gel could
take 6 - 7 hours. Run minigels at 15 mA. It should take about an hour to run.
9
SDS-PAGE
5. Stain and destain the gel
Stain gel in 10 volumes of Coommassie Blue Stain for 1 hour at room temperature. Save staining
solution. Re-use solution for 1 month to 1 year. This step may go overnight.
Destain gel in 10 volumes of 1X destain for 30 minutes at room temperature.
After the destain the gel, gel is ready to visualize or drying the gel
10