Lab: Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)

PAGE
Polyacrylamide gel electrophoresis (PAGE) is probably the most common analytical
technique used to separate and characterize proteins. A solution of acrylamide and
bisacrylamide is polymerized. Acrylamide alone forms linear polymers. The bisacrylamide
introduces crosslinks between polyacrylamide chains. The 'pore size' is determined by the
ratio of acrylamide to bisacrylamide, and by the concentration of acrylamide. A high ratio of
bisacrylamide to acrylamide and a high acrylamide concentration cause low electrophoretic
mobility. Polymerization of acrylamide and bisacrylamide monomers is induced by
ammonium persulfate (APS), which spontaneously decomposes to form free radicals.
TEMED, a free radical stabilizer, is generally included to promote polymerization.

SDS-PAGE
Sodium dodecyl sulfate (SDS) is an amphipathic detergent. It has an anionic headgroup and a
lipophilic tail. It binds non-covalently to proteins, with a stoichiometry of around one SDS
molecule per two amino acids. SDS causes proteins to denature and dissassociate from each
other (excluding covalent cross-linking). It also confers negative charge. In the presence of
SDS, the intrinsic charge of a protein is masked. During SDS PAGE, all proteins migrate
toward the anode (the positively charged electrode). SDS-treated proteins have very similar
charge-to-mass ratios, and similar shapes. During PAGE, the rate of migration of SDS-treated
proteins is effectively determined by molecular weight.

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Below is an example of the procedure for performing discontinuous SDS-PAGE with a 14%
separating gel and a 5% stacking gel.

Materials

PAGE Rigs including glass plates (10 x 20 cm), spacers, comb, and clamps

Power supply

Protein sample

Bio-Rad Laemmli Sample Buffer (contains SDS and either sucrose or glycerol)

2-Mercaptoethanol (reduces disulfide bonds, disrupts protein cross-links)

Molecular Weight Markers (already prepared in sample buffer)

Gel Cassette Assembly (Bio-Rad Mini Protean 3)

 Clean and completely dry the glass plates, combs, and any other pertinent materials.

 Place a short plate on top of a spacer plate. Insert both plates into the green casting
frame on a flat surface. Be sure that the "legs" of the casting frame are down. Clamp
the casting frame and check that the plates are level on the bottom.

 Put the casting frame into the casting stand.

Preparation of the Gel

 Combine all reagents except the TEMED for the 14% separating gel.

14% Separating Gel Components (4.195 mL)

1.184 milliliters deionized water

1.96 milliliters 30% acrylamide/Bisacrylamide (Warning: Acrylamide is a

neurotoxin. Use gloves, do not ingest.)

1.0 milliliters 1.5 M Tris, pH 8.8

21 microliters 20% SDS

12 microliters 10% ammonium persulfate

18 microliters TEMED, pH 8.9

  When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and
transfer the separating gel solution between the glass plates in the casting chamber to
about 3/4 inch below the short plate.

 A small layer of butanol can be added on top of the gel prior to polymerization to
straighten the level of the gel and remove unwanted air bubbles that may be present.
Butanol will not mix with the aqueous separating gel solution. Once the gel has
polymerized, the butanol can be removed by absorption with Kimwipes or filter
paper. Rinse the top layer of the gel with dI water prior to pouring the stacking gel.

 Insert the well forming comb into the opening between the glass plates.

 Combine all reagents except the TEMED for the 5% stacking gel.

5.1% Stacking Gel (2.484 mL)

0.4 milliliters deionized water

1.8 milliliters 30% acrylamide/Bisacrylamide

0.25 milliliters 0.5 M Tris, pH 6.8

12 microliters 20% SDS

17 microliters 10% ammonium persulfate

5 microliters TEMED

 When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and
transfer the stacking gel solution between the glass plates in the casting chamber.

 Both the separating and stacking gels should polymerize within six minutes.

 Once the stacking gel has polymerized, the comb can be gently removed. The
polymerized gel between the short plate and spacer plate forms the "gel cassette".

Sample Preparation

 Place some water in a 600 mL beaker and leave on a hot plate to boil. (This can take
15 minutes or more.)

 Meanwhile, add 50 mL of 2-mercaptoethanol to 950 mL of Laemmli sample buffer.

 Combine 10 mL of each protein sample with 20 mL of Laemmli sample buffer plus 2-

mercaptoethanol in microcentrifuge tubes.

 In separate tubes, aliquot 10 mL of MW marker. (MW markers are already prepared

in Laemmli sample buffer.)
  Boil the samples for no more than 5 minutes to fully denature the proteins. Leave the
samples at room temperature until ready to load onto the gel.

Electrophoresis

 Remove the gel cassette from the casting stand and place it in the electrode assembly
with the short plate on the inside.

 Slide the electrode assembly (with the gel cassette) into the clamping frame. Press
down on the electrode assembly while clamping the frame to secure the electrode
assembly. This step is important to minimize potential leakage during the
electrophoresis experiment.

 Pour some 1X electrophoresis buffer into the opening of the casting frame between
the gel cassettes. Add enough buffer to fill the wells of the gel. Use a gel loading tip
to pipette some buffer into each well to ensure cleanliness.

 When all wells are sufficiently cleaned, slowly pipette no more than 30 mL of
denatured sample or MW marker into each well. A yellow guide can be placed on top
of the electrode assembly to aid in loading the gel.
 When the gel has been loaded, lower the clamping frame into the electrophoresis tank.

 Fill the region outside of the frame with 1X electrophoresis buffer.

 Cover the tank with the lid aligning the electrodes (black or red) appropriately.

 Connect the electrophoresis tank to the power supply.

 Allow the samples to run at 30 mA until the dye front reaches the bottom of the gel.
This can take as long as 1 hour.

 When electrophoresis is complete, turn off the power supply and disassemble the
apparatus.

Point to Ponder: SDS-PAGE has a top layer of stacking gel and a bottom layer of separating
gel with different pH values of 0.5M Tris-HCl. The stacking was 6.8 and the separating gel
was 8.8. What about this pH change makes the gels different? What effect does this have on
the proteins that we run?

Answer:

 The stacking gel concentrates proteins loaded into the sample wells so that they are
resolved as a unified "line" once they enter the stacking gel. The reason for the lower
pH is that this "lower ionic strength implies higher electrical resistance and
consequently a higher electric field, provoking the faster movement of the proteins
and of every other charged particle in the gel. Such a high electric field coupled with
the glycine in the running buffer (that will not go into the resolving gel due to the pH),
helps to clean the sample from the Cl- ions from the Tris-HCl buffer. That concludes
in a stack of clean, denatured, and equally charged proteins in the boundary with the
resolving gel; this will account for a good quality PAGE."
 Bassically, in the stacking gel the pore size is larger so it gives a chance for the larger
proteins and smaller proteins to equalize with each other.
 "As the proteins are denatured and linearized by heating, SDS and dithiothreitol (if
there is any di-sulfide bond), the proteins are loaded onto the wells on the stacking
gel. The denatured proteins have a uniform mass to negative charge ratio. Since the
current run from negative (top) to positive (bottom), the proteins move toward the
bottom. When the electricity is turned on, the proteins and Tris-glycine enter the
stacking gel. In stacking gel with pH 6.8, the N-terminal amino group of the proteins
and amino acids are protonated at equilibrium which makes them less negative. The
average electrophoretic mobility is very slow. A Gly-chloride ion boundary is formed
since glycine moves slower than chloride ion. However, glycine still runs slightly
faster than other proteins. As a result, the glycine keeps pushing the protein towards
the chloride ion. In other words, the proteins are trapped between glycine and chloride
ion. The proteins form a very tight band inside the stacking gel.

 Once the protein reaches the resolving gel, the pH changes from 6.8 to 8.8 and the
pores are smaller. As pH increases, the N-terminal amino groups are deprotonated.
Amino acids and proteins are more negatively charged at equilibrium than in stacking
gel. As a result, glycine moves faster than proteins. Glycine and chloride ions move
ahead of the proteins. As the pores are smaller in the resolving gel, the size of the
protein determines the mobility (speed). So the smaller the protein is, the faster it will
reach the bottom."

 The other major difference between the two is the amount of acrylamide in the upper
(stacking) gel - it's generally around 4%, while the lower (resolving) gel can vary
from 6 or 8% to 20%, depending on the size of the protein(s) you're looking for.

 When you load your samples in the wells at the top of the gel, then start the current,
not all of the sample enters the gel at the same time. The purpose of the stacking gel,
with its lower pH and low acrylamide percentage, is to "stack" all of the proteins in
your sample into as narrow of a band as possible, so that they all enter the resolving
gel at essentially the same time. If you didn't have that stacking gel, your band(s) of
interest could be very diffuse, and may not match up with your molecular weight
marker.