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X-tal protocols

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Protein Detergent Methods Euipment Screen Drops Reference

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Background.

The anionic detergent sodium dodecyl sulfate (SDS) dissociates proteins in their constituent polypeptide chains. Monomeric SDS binds tightly to most proteins at about 1.4 mg SDS/mg protein. Combined treatment with a disulfide reducing agent (2-mercapto ethanol or dithiothreitol) completely unfolds the protein.

Polyacrylamide gel electrophoresis in the presence of SDS separates the polypeptide chains according to their molecular weight. Thus the molecular weight of the polypeptide chains of a given protein can be determined by comparing their electrophoretic mobility on SDS gels to the mobility of marker proteins with polypeptide chains of known molecular weights.

The most popular electrophoretic method is the SDS-PAGE system developed by Laemmli [Leammli, 1970]. This is a discontinuous system consisting of two gels: a stacking and a separating gel. The samples are prepared in low conductivity buffer (60 mM Tris-Cl, pH=6.8) and are loaded between the higher conductivity electrode buffer (25 mM Tris, 192 mM glycine, pH=8.3) and the stacking gel (125 mM Tris-Cl, pH=6.8). When power is applied, a moving boundary region is rapidly formed with the highly mobile chloride ions in the front and the relatively slow glycinate ions in the rear. A localized high voltage gradient forms between the fronts, causing the SDS-protein complex to condense into a very narrow region and migrate between the chloride and glycinate phases. The total amount of SDS has a considerable influence on resolution. High concentrations of SDS can lead to broadening and spreading of protein bands. At the interface of the stacking and separating gel the SDS-protein complex experience a sharp increase in retardation due to the restrictive pore size of the resolving gel. The glycinate ions overtake the proteins, which then move in a space of uniform pH formed by the Tris and the glycine. [Garfin, 1990]

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The Gel.

The separating gel.

Clean the glass plates, spacers and comb with detergent, rinse with water and dry. Wipe the glass plates with ethanol.
Assemble the glass plate sandwich.
Depending on the gel system used, you might have to change the volumes.

Wear gloves while working with acrylamide. Prepare the separating gel solution except ammonium persulfate and TEMED in a vacuum flask. De-aerate for 1 min. under vacuum. After adding the ammonium persulfate and TEMED mix the solution gently.

separating gel 10 % 15 %
range16-70 kD12-45 kD
water1600 µl939 µl
1.5 M Tris pH 8.81000 µl1000 µl
30% Acrylamide1333 µl2000 µl
10% SDS40 µl40 µl
20% Ammonium persulfate14 µl14 µl
TEMED7 µl7 µl

With a pipet fill the separating gel solution between the glass plate sandwich along the edge of one of the spacers, until the height of the solution is 1.0 cm below the comb. Immediately overlay the solution with water saturated 2-butanol or isopropanol to exclude air and to obtain an even interface between the gels.
Allow the gel to polymerize for 45 min. The gel is polymerized when a sharp interface is visible below the overlay.

The stacking gel.

Prepare the stacking gel just before using the gel, to maintain the ion discontinuities at the interface between the two gels. Mix the ingredients and de-aerate before adding the ammonium persulfate and TEMED.

stacking gel
water1475 µl
0.5 M Tris pH 6.8625 µl
30% Acrylamide 335 µl
10% SDS25 µl
20% Ammonium persulfate10 µl
TEMED3 µl

Remove the solution from the top of the gel, rinse with water and dry the area above the gel carefully with filter paper. Fill the stacking gel solution on top of the separating gel. Place the well forming comb in position, being careful not to trap air bubbles under the teeth. Visible polymerization of the gel should occur within 20 min.

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Sample Preparation.

2* Sample buffer:
0.5 M Tris pH 6.82.4 ml
10 % (w/v) SDS4.0 ml
0.2 % Bromophenolblue(w/v in water)0.6 ml
Glycerol2.0 ml

Prepare the 2* SDS-reducing sample buffer by adding 100 µl 2-mercaptoethanol to each 0.9 ml 2* Sample buffer. Dilute the sample (10 µl) with an equal volume of 2* SDS-reducing sample buffer. Heat the samples with cytoplasmic proteins for 3 min at 95°C. Hydrophobic (membrane) proteins are incubate 15 min at 37°C ( or 10 min at 60°C) to avoid aggregation.

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Electrophoresis.

Dilute the 5* running buffer with 4 volumes of water.

5 * Running buffer (for 1 liter)
Tris15 g
Glycine72 g
SDS5 g

Assemble the electrophoresis cell, remove tubing, clamps and comb. Fill the upper and lower reservoir with electrode buffer. Load the samples into the wells in the stacking gel. Connect the electrophoresis unit to the power supply. The lower electrode is the anode (+) and the upper is the cathode (-). Start with a low voltage (70 volt) and increase to a higher (200 volt) when the samples entered the stacking gel, continue the electrophoresis until the blue dye has reached the bottom of the gel.

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Detection of Proteins in Gels.


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