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HBC 1019 Biochemistry 1 Trimester 1, 2012/2013

Practical 5: Protein Separation using Gel Electrophoresis: SDS-PAGE

IntroductionElectrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.

SDS-PAGE stands for Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and is a method useful in separation and identification of proteins according to their size. Since different proteins with similar molecular weights may migrate differently due to their differences in secondary, tertiary or quaternary structure, SDS which is an anionic detergent is used in SDS-PAGE to reduce proteins to their primary (linearized) structure by breaking up the complex structure into their polypeptide subunits in the process of denaturation of proteins. It then coats them with uniform negative charges by the process called denaturation.

Generally, the procedures involve in SDS-PAGE are:1) Making a gel and assembling the gel apparatus2) Mixing protein samples with sample buffer containing SDS and heat the mixture at high temperature3) Loading samples and running the electrophoresis4) Fixing and staining the separated proteins.

Generally the sample is run in a support matrix which is polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Polyacrylamide is easier to handle when it is made at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.

Materials, Reagents and Equipments

I) Mini-Protean Electrophoresis system Components

Spacer plate The Spacer Plate is the taller glass plate with gel spacers permanently bonded. Spacer Plates are available in 0.5 mm, 0.75 mm, 1.0 mm, and 1.5 mm thicknesses, which are marked directly on each Spacer Plate.

Short plate The Short Plate is the shorter, flat glass plate that combines with the Spacer Plate to form the gel cassette sandwich.

Casting frame The Casting Frame, when placed on the benchtop, evenly aligns and secures the Spacer Plate and the Short Plate together to form the gel cassette sandwich prior to casting.

Gel cassette assembly One Casting Frame, a Spacer Plate, and a Short Plate form one Gel Cassette Assembly.

Casting stand The Casting Stand secures the Gel Cassette Assembly during gel casting. It contains pressure levers that seal the Gel Cassette Assembly against the casting gaskets.

Gel cassette sandwich A Spacer Plate and Short Plate with polymerized gel form a Gel Cassette Sandwich after casting.

Combs A selection of molded combs is available.

Buffer dam The molded, one-piece buffer dam is used when running only one gel.

Electrode assembly The Electrode Assembly holds the Gel Cassette Sandwich. It houses the sealing gasket, the upper and lower electrodes and the connecting

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banana plugs. The anode (lower electrode) banana plug is identified with a red marker and the cathode (upper electrode) banana plug with a black marker.

Clamping frame The Clamping Frame holds the Electrode Assembly and Gel Cassette Sandwich in place. Its pressure plates and closure cams seal the Gel Cassette Sandwich against U-shaped gaskets on the Electrode Assembly to form the inner buffer chamber.

Inner chamber The Electrode Assembly, two Gel Cassette Sandwiches or one gel cassette sandwich and a buffer dam, and the Clamping Frame form the Inner Chamber.

Mini tank and lid The Mini Tank and Lid combine to fully enclose the inner chamber during electrophoresis. The lid cannot be removed without disrupting the electrical circuit.

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II) Sample loading buffer (Laemmli Loading dye) 3X stock1M Tris-Cl, pH6.8 2.4ml20% SDS 3.0mlGlycerol 3.0mlβ-mercaptoethanol 1.6mlBromophenol blue 0.6gTotal volume is 10ml. Store at 4oC.

III) 10X Running Buffer (also called Laemmli Buffer)Tris Base (15g/l)Glycine (72g/l)SDS (1%)Make to 1L with dH2O

IV) 1 M Tris-HCl, pH8.8V) 1 M Tris-HCl, pH6.8VI) 10% APS (ammonium persulphate)VII) TEMED (N,N,N',N'-Tetramethylethylenediamine)VIII) 0.25% (w/v) Coomassie Brilliant Blue R 250 in methanol-water-glacial acetic acid (5-5-1), filtered

immediately before useVIIII) Samples (from Practical 4)

Methods

I) Gel cassette and casting stand assembly

Note: Ensure the casting stand, casting frames and glass plates are clean and dry before setting up the casting stand assembly.

1. Place the Casting Frame upright with the pressure cams in the open position and facing on a flat surface.

2. Select a Spacer plate of the desired gel thickness and place a Short Plate on top of it (see Figure 4a)

3. Orient the Spacer Plate so that the labeling is “up”. Slide the two glass plates into the Casting Frame, keeping the Short Plate facing the front of the frame (side with pressure cams) (see Figure 4b).

Note: Ensure both plates are flush on a level surface and labeling on the Spacer Plate is oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly.

4. When the glass are up in place, engage the pressure cams to secure the glass cassette sandwich in the Casting Frame (see Figure 4c). Check that both plates are flush at the bottom.

5. Engage the spring loaded lever and place the gel cassette assembly on the gray casting stand gasket. Insure the horizontal ribs of the back of the Casting Frame are flush against the face of the Casting Stand and the glass plates are perpendicular to the level surface. The lever pushes the Spacer Place down against the gray rubber gasket (see Figure 4d).

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II) Gel Casting

Discontinuous Polyacrylamide gels1. Place a comb completely into the assembled gel cassette. Mark the glass plate 1cm below the

comb teeth. This is the level to which the resolving gel is poured. Remove the comb.2. Prepare the resolving gel monomer solution by combining all reagents as follow:

Reagents Percentage of gel: 12.5%37.5:1% w/v acrylamide:bisacrylamide 4.1 ml1.0M Tris-Cl pH8.8 4.0 ml20% SDS 50.7 uldH2O 1.73 ml

3. Add APS and TEMED to monomer solution and pour to the mark using a glass or disposable plastics pipette.

Reagents Volume10% APS 48.0 ulTEMED 6.7 ul

4. Immediately overlay the monomer solution with water or butanol.5. Allow the gel to polymerize for 45 minutes to 1 hour. Rinse the gel surface completely with

distilled water. Do not leave the alcohol overlay on the gel for more than 1 hour because it will dehydrate the top of the gel.

Note: At this point the resolving gel can be stored at room temperature overnight. Add 5 ml of 1:4 dilution of 1.5 M Tris-HCl, pH8.8 buffer (for Laemmli System) to the resolving gel to keep it hydrated. If using another buffer system, add 5ml of 1x resolving gel buffer to the resolving gel surface for storage.

6. Prepare the stacking gel monomer solution as follow:Reagents Percentage of gel: 6.0%37.5:1% w/v acrylamide:bisacrylamide 1.0 ml1.0M Tris-Cl pH6.8 630 ul20% SDS 25 uldH2O 3.0 ml

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Note: Use a 4% of stacking gel if the resolving gel percentage is <10% and a 6% of stacking gel (easier to handle) if the resolving gel percentage is >10%

7. Add APS and TEMED to the stacking gel monomer solution and pour the solution between the glass plates. Continue to pour until the top of the short plate is reached.

Reagents Volume10% APS 48.0 ulTEMED 6.7 ul

8. Insert the comb between the spacers starting at the top of the Spacer Plate, making sure that the tabs at the ends of each comb are guided between the spacers.

9. Allow the stacking gel to polymerize for 30-45 minutes.10. Gently remove the comb and rinse the wells thoroughly with distilled water or running buffer.

III) Sample loading

1. Remove the Gel Cassette Assemblies from the Casting Stand. Rotate the cams of the Casting Frames inward to release the Gel Cassette Sandwich (see Figure 5a).

2. Place a Gel Cassette Sandwich into the slots at the bottom of each side of the Electrode Assembly. Be sure the Short Plate of the Gel Cassette Sandwich faces inward toward the notches of the U-shaped gaskets (see Figure 5b).

3. Lift the Gel Cassette Sandwich into place against the green gaskets and slide into the Clamping Frame (see Figure 5c).

4. Press down on the Inner Chamber and to insure a proper seal of the short plate against the notch on the U-shaped gasket. (see Figure 5d). Short plate must align with notch n gasket.

Note: Gently pressing the top of the Electrode Assembly while closing the Clamping Frame cams forces the top of the Short Plate on each Gel Cassette Sandwich to seat against the rubber gasket properly and prevents leaking.

5. Lower the Inner Chamber Assembly into the Mini Tank. Fill the inner chamber with ~125 ml of running buffer until the level reaches the halfway between the tops of the taller and shorter glass plates of the gel Cassettes.

Note: Do not overfill the Inner Chamber Assembly. Excess buffer will cause the siphoning of buffer into the lower chamber which can result in buffer loss and interruption of electrophoresis.

6. Add ~200 ml of running buffer to the Mini Tank (lower buffer chamber)

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7. Mix your protein 4:1 with sample buffer and heat your sample by boiling for 5-10 minutes. Load the samples into the wells with a pipette using gel loading tips.

IV) Electrophoresis1. Apply power and begin electrophoresis at room temperature; 200 V constant until the

bromophenol blue is just off (approximately 50-60 minutes).

V) Visualization of Protein Separation1. After electrophoresis, transfer the gel into a washing tray and rinse with dH2O twice.2. Place the gel in at least 10 volumes of Coomasie Blue staining solution for 2-4 hours. Rock

gently to distribute the dye evenly over the gel. 3. At the conclusion of the staining, wash the gels with several changes of water. 4. Place the gels into a solution of 10% acetic acid for at least 1 hour.

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5. If the background is still deeply stained at the end of the hour, move the gels to fresh 7% acetic acid as often as necessary.

6. Photograph the gels or analyze the gels spectrophotometrically

Questions1. What is the principle used in electrophoresis?2. What is the function of SDS in this experiment?3. In your opinion, which protein molecules are able to move faster in the gel: large macromolecules

or small molecules? Where do you think the positions of these molecules in the gel? 4. You are given a solution which contains a mixture of the six following proteins: Carbonic Albumin

from Bovine 66 kDa, Anhydrase from Bovine 29 kDa, β-Galactosidase from E. coli 116 kDa, Albumin from egg 45 kDa, Phosphorylase B from Rabbit 97.4 kDa, Myosin from Rabbit muscle 200 kDa. Draw a schematic representation of the protein separation in a SDS-PAGE.

5. How would you prepare a 1.5 M of Tris-HCl, pH8.8 solution (Given the molecular weight of Tris-HCl is 157.59)?

6. How would you prepare a 20% (w/v) SDS?7. How would you prepare 1 litre of a running buffer which contains the following properties:

25mM Tris-HCl200mM Glycine 0.1% (w/v) SDS

(Use the 1.5M Tris-HCl and 20% SDS solutions prepared in Question 5 and 6) The molecular weight for Glycine is 75.07.

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