SDS-PAGE

• Sodium Dodecylsulfate Polyacrylamide Gelelectrophoresis
• analysis of purity and molecular mass of proteins
• separation according to their size only

Location:

Arnimallee 22, E030

Responsible person: 

Michael Tully, Arnimallee 22, E030, michael.tully@fu-berlin.de

Stefanie Wedepohl, Arnimallee 22, E001, stefanie.wedepohl@fu-berlin.de

Assay principle

SDS-PAGE is a method to separate and analyze proteins in the range of 5-250 kDa mainly by size. This is achieved by using SDS, a surfactant that binds to proteins and thereby masks their individual charge, resulting in equally distributed negative charge on the protein surface. The protein's secondary structure can be further eliminated by heat denaturation and/or reduction of the disulfide bonds. Proteins are loaded onto a polyacrylamide gel matrix and exposed to an electric field, where they migrate due to their charges. Smaller proteins migrate in the gel matrix faster than larger proteins. Because of the uniform negative charge mediated by SDS, separation is therefore dependent only on the size of the proteins and the gel mesh.

Gel mesh sizes depending on the acrylamide concentration and buffer systems can be chosen according to the desired separation range.

For detailed information about One-dimensional SDS gel electrophoresis of proteins see the Current Protocols. A protocol of the classical Laemmli method that is usually run in our lab is given below.

It is necessary to include a standard molecular weight marker in each run (as in the example in the image above) to be able to compare to the protein of interest. The samples should all have the same salt concentration, buffer and volume to avoid distortions in the gel. Sample overloading also leads to distortions, so you'd start at 2µg max/lane for a pure protein (examples and troubleshooting at the Rice University Hall of Shame)

After running, the gel is transferred into staining solutions to visualize protein bands. The most common staining methods include Coomassie brilliant blue R250 (detection range: 300-1000 ng per band) and silver staining (1-10 ng per band).

After destaining, the bands can be documented via the Gel-Doc system or transfered to membranes for western blot analysis.

Discontinuous Tris-Glycine SDS-PAGE (classical Laemmli system) with Coomassie staining

10x SDS-PAGE Sample buffer:

• mix 10 mL 1.5M Tris (pH6.8), 6mL 20% SDS, 30% glycerol, 15 mL β-mercaptoethanol and 1.8 mg bromophenol blue.
• adjust volume to 100mL with H2O
• aliquot in 10mL stock solution and store at -20°C
• store working solution at 4°C

10x SDS PAGE running buffer:

• dissolve 10g SDS, 30.3g Tris and 144.1g glycin in 800 mL H₂O
• adjust volume to 1 L with H₂O
• store at r.t.

other stock solutions needed:

• 1.5M Tris HCl pH 8.8 in H₂O
• 10% SDS in H₂O
• 10% APS (ammonium persulfate) in H₂O
• TEMED (Tetramethylethylenediamine)
• Rotiphorese 30 (37,5:1 acrylamide : bisacrylamide mixture)

Coomassie Blue staining solution:

• dissolve 2.5g  Coomassie brilliant blue R-250 in a mixture of 450 mL methanol, 100 mL acetic acid and 400 mL H₂O
• adjust volume to 1 L with H₂O
• store at r.t.

Coomassie Blue destaining solution

• mix 450 mL methanol, 100 mL acetic acid and 400 mL H₂O
• adjust volume to 1 L with H₂O
• store at r.t.

Procedure:

• use stock solutions as prepared above. APS aliquots are stored in the freezer, Rotiphorese 30 (37,5:1 acrylamide : bisacrylamide mixture) and TEMED (Tetramethylethylenediamine) should be found in the fridge.
  
• Assemble the glass plates of the gel chamber in the gel preparation stand and check for leaks with water. Mark the height of the separation gel using the comb.
• mix the following solutions (don't add the TEMED until ready to polymerize)
• Recipes for 2 separating gels of either 7.5%, 10% or 13% (scale up as needed)

• start the polymerization by addition of TEMED and immediately pour the solution between the plates until the marking.
• add some isopropanol to the surface of the gel to break bubbles
• wait until polymerized, then remove the isopropanol, rinse with water and dry with filter paper
• prepare mixture for stacking gels:

• add TEMED when ready for polymerization
• pour mixture on top of the separating gel
• immediately insert comb
  • make sure all pockets are closed tight, i.e. no air bubbles trapped
• wait until polymerized (about 20 minutes)
• use immediately or store under conditions where it doesn't dry out (in a plastic bag in the fridge)

prepare samples:

• for combs with 10 pockets use 20µL of protein solution (about 2µg individual protein, more possible if it is a mixture)
• mix with 6.6µL of 4x sample buffer (in the fume hood if it contains ß-mercaptoethanol)
• heat to 96-100°C for 10 minutes (be careful, the lid of the eppendorf tubes might pop)

electrophoresis run:

• carefully remove combs (rinse pockets with water if desired)
• assemble gels in electrophoresis chamber
• add 1x running buffer: pour into the inner chamber and let it overflow to the outer chamber either fully or half. make sure that there is about 1cm above the pockets on the inside chamber
• apply samples to gel pockets using a hamilton syringe
• apply 3-5µL of ready to use protein standard.
• close the chamber and connect to power supply
• run at 100-200V until the blue dye that marks the front reaches the end of the gel (see if the dye comes out at the bottom)
• stop the run and disassemble /rinse everything with water

Coomassie staining:

• disassemble glass plates and mark the bottom right corner
• pinch off stacking gel and discard
• rinse the gel with water into a glass container
• remove water and add coomassie staining solution
• stain for about 20 minutes or longer on the shaker
• discard staining solution and add destaining solution
• replace staining solution every 20 minutes until protein bands are clearly visible above background.

Manual for the electrophoresis chamber

Link to gel documentation system