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]
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 % range 16-70 kD 12-45 kD water 1600 µl 939 µl 1.5 M Tris pH 8.8 1000 µl 1000 µl 30% Acrylamide 1333 µl 2000 µl 10% SDS 40 µl 40 µl 20% Ammonium persulfate 14 µl 14 µl TEMED 7 µl 7 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 water 1475 µl 0.5 M Tris pH 6.8 625 µl 30% Acrylamide 335 µl 10% SDS 25 µl 20% Ammonium persulfate 10 µl TEMED 3 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.
2* Sample buffer: 0.5 M Tris pH 6.8 2.4 ml 10 % (w/v) SDS 4.0 ml 0.2 % Bromophenolblue(w/v in water) 0.6 ml Glycerol 2.0 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.
Dilute the 5* running buffer with 4 volumes of water.
5 * Running buffer (for 1 liter) Tris 15 g Glycine 72 g SDS 5 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.
|Staining solution (5/5/1):|
|Coomassie Brilliant Blue R-250||0.1 % (w/v)|
|Methanol||45.5 % (v/v)|
|Acetic acid||9.0 % (v/v)|
Filter the solution before use. Soak the gel in an excess of staining solution for 1 hour.
Destain with destain solution, with a Kleenex Tissue (to absorb the CCB-R250) until the background is clear.
|Methanol||5.0 % (v/v)|
|Acetic acid||7.0 % (v/v)|
Fixation for at least 1 h in 12 % (W/V) TCA
|Coomassie Brilliant Blue G-250||0.1 % (w/v)|
|Phosphoric acid||2.0 % (w/v)|
|Ammonium sulfate||6.0 % (w/v)|
The staining solution should be prepared in the following sequence:
only after complete solubilization of the ammonium sulfate in the acid
the aliquot volume of a stock solution of CBB G-250 (1 g/ 20 ml water)
should be added.
The staining solution should never be passed through a filter.
The staining solution should be shaken prior to use for even
distribution of the colloidal particles.
Stain until the bands are clearly visible (2 hours or o/n). Destain with destain solution.