BIOLS 315

Biochemistry Lab. No. 3

Separation and Characterization of Proteins Using SDS –

Polyacrylamide Gel Electrophoresis Technique

Introduction
Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) is a low-
cost, reproducible, and rapid method for quantifying, comparing, and characterizing
proteins. This method separates proteins based primarily on their molecular weights.
SDS binds along the polypeptide chain, and the length of the reduced SDS-protein
complex is proportional to its molecular weight. The relative ease of performing
SDS-Page along with its many applications has made it an important analytical
technique in many fields of research.

Sodium dodecyl sulfate (SDS) is an anionic detergent that very effectively disrupts
the quaternary structure of most multimeric proteins. Many SDS molecules bind
tightly to the subunits thereby obscuring the original charge on the protein (Fig.1a).
Thus, during electrophoresis, all SDS-protein complexes migrate toward the positive
pole with about the same charge/mass ratio. If electrophoresis is carried out on
polyacrylamide gels, mobility of SDS-protein complex will depend almost
exclusively on its size. In effect, SDS gel electrophoresis is gel filtration with an
electric field as the driving force, instead of bulk solution flow. Thus, a standard curve
can be prepared using proteins with known monomer molecular weights (Fig.1b). The
MW of an unknown can then be easily determined. SDS gel electrophoresis can be
performed on nonhomogeneous preparations if it is possible to locate the position of
the protein of interest (e.g., by some specific stain for that protein, or one of its
reaction products if the protein is an enzyme).

Our description of SDS-PAGE will include protocols for linear slab gels, i.e. gels
formed as slabs between two sheets of supporting glass. Slab gels have become more
widely used than tube gels (formed with glass tube supports), since many samples can
be run on the same gel, thereby providing uniformity during polymerization, staining,
and destaining. For most analytical applications, the mini slab gel has gained
considerable popularity due to the increased resolution and reduced amounts of time
and materials required for electrophoresis. The experimental procedures and reagents
in this volume have been calculated for use with a mini-gel system; however, all
procedures should be readily adaptable to other systems.

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Equipment
Minigel apparatus
Power supply (capacity 200v, 500mA)
Boiling water bath
Eppendrof tubes
Gel dryer and high vacuum pump
100 ml side arm flask with stopper
Vortex
Pipettes and micropipettes

Preparation of SDS-Polyacrylamide Gels

A Separating Gel

Reagents:
1 Separating gel buffer pH 8.8 (0.375M Tris-HCl + 0.1% SDS).

2 Acrylamide stock solution (30% (w/v) acrylamide, 0.8% (w/v) bis-

acrylamide).
Caution:
Unpolymerized acrylamide is a skin irritant and a neurotoxin. Always
handle with gloves.
3 10% ammonium persulfate (freshly prepared).
4 TEMED (N, N, N, N – tetramethylene-ethylenediamine).

Procedure
1 Assemble your slab gel apparatus with the glass sandwich set.
2 Prepare a separating gel from the ingredients listed above.
3 First, add reagents 1 and 2 to a side arm flask in volumes 5.95 ml and 4.0 ml
respectively. Stopper the flask and attach to a vacuum pump. Turn on the
vacuum and degas the solution for approximately 10 min. During this period,
gently swirls the solution in the flask.
4 Turn off the vacuum, open the flask and add 75 l of 10% ammonium
persulfate and 10 l of TEMED to the solution. Mix by swirling gently.
Work rapidly at this point because polymerization will be under way.
5 Carefully introduce this solution into the gel sandwich using a pipette. Pipe
solution so that it descends along a spacer. This minimizes the possibility of
air bubbles becoming trapped within the gel.
6 When the appropriate amount of separating gel solution has been added (leave
about 1.5 cm from top of front plate for stacking gel, gently layer about 1 cm
of water on top of the separating gel solution. This keeps the gel surface flat
and prevents the dehydration of the gel.
7 Allow gel to polymerize (30-60 min) without disturbance.

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 Stacking Gel

Reagents:
1 Stacking gel buffer pH 6.8 (0.125M Tris-HCl + 0.1% SDS).
2 Acrylamide stock solution (30% (w/v) acrylamide, 0.8% bis-
acrylamide).
3 10% ammonium persulfate (freshly prepared).
4 TEMED

Procedure
1 Pour off water covering the separating gel. The small droplets remaining will
not disturb the stacking gel.
2 Prepare a stacking gel from the above listed ingredients.
3 First, add reagents 1 and 2 to a side arm flask in volumes 4.35 ml and 0.65 ml
respectively. Degas the solution.
4 Add 75 l 10% ammonium persulfate and 10l TEMED. Mix gently.
5 Pipette stacking gel solution onto separating gel until solution reaches top of
front plate.
6 Carefully insert comb into gel sandwich until bottom of teeth reach top of
front plate. Be sure no bubbles are trapped on ends of teeth. Tilting the comb
at a slight angle is helpful for insertion without trapping air bubbles.
7 Allow stacking gel to polymerize (about 30 min) prior to use.

Preparing, Loading Samples and Running the Gel

Reagents:
Protein standard (1 mg/ml of sample buffer)
Sample buffer pH 6.7 (0.01M Tris-HCl + 1% SDS)
Electrophoresis running buffer pH 8.3 (Tris-Glycine + SDS)
0.25% Bromophenol blue in 40% sucrose (w/v)
2- Mercaptoethanol
Micropipettes

Procedure
1 Remove the combs from prepared gels by gently lifting the combs from the
chamber. Rinse the wells with running buffer.
2 Take 100 l of your standard or sample in an Eppendrof tube. Add 5 l 2-
Mercaptoethanol, shake well and boil in a water bath for 5-7 min.
3 Cool the solution and add 100 l Bromophenol blue dye and mix well.
4 Using a micropipette or a syringe, add 5 l of your treated sample to the
bottom of a well.
5 Remove the gel from its casting stand and assemble it into the appropriate slab
unit for running the electrophoresis.
6 Pour a sufficient quantity of running buffer into the chamber of the
electrophoresis apparatus until the bottom of the gel is immersed in buffer, and

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 the top is covered. Be careful not to disturb the samples in the wells when
adding buffer to the upper chamber.
7 Assemble the top of the electrophoresis apparatus and connect the system to an
appropriate power source. Be sure that the …… (x) is connected to the upper
buffer chamber.
8 Turn on the power supply and run the gel at 20 mA constant current per 1.5
mm gel. For example, if two gels are running, each having 1.5 mm spacers,
the current should be adjusted to 40 mA. One gel with 1.5 mm spacers should
be run at 20 mA, while a gel with 0.75 mm spacers should be run at 10 mA.
9 When the tracking dye reaches the separating gel layer, increase the current to
30 mA per 1.5 mm gel.
10 Continue applying the current until the tracking dye reaches the bottom of the
separating gel layer.

11 Turn off and disconnect the power supply. Disassemble the gel apparatus and
remove the glass sandwich containing the gel. Carefully remove a spacer, and
prepare gently the gel plates. The gel will stick to one of the plates.

Staining a Gel with Coomassie Blue and Destaining

Reagents:
Coomassie blue R-250
Destain solution
Glass dishes

Procedure

1 Wearing gloves to prevent transfer of finger prints to the gel, pick up the gel
and transfer it to a glass dish (taking care not to tear the gel) containing
enough volume of Coomassie stain.
2 Agitate for a few seconds, and cover the dish with a lid.
3 Leave overnight.
4 Pour out the stain the next day and transfer the gel to another glass dish
containing Coomassie Gel Destain solution. Agitate and leave one hour.
Strong bands are visible immediately. To destain completely, change destain
solution and leave overnight.

Drying a Gel

Reagents:
Whatman 3 mm paper
Gel dryer apparatus supplied with a vacuum pump

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Procedure
1 Place gel upside down on a Whatman paper. The gel should stick to the paper.
2 Cover the front of the gel with an acetate sheet or plastic wrap, taking care not
to trap air bubbles, which can lead to gel cracking.
3 Place Whatman paper on gel dryer, turn on heat at 80oC for 20 min and
suction, then cover with a sealing gasket. After 20 min, reduce the
temperature to 60oC for 15-20 min.
4 Plot the relative mobility of each protein against the log of its molecular
weight.

Relative mobility is the term used for the ratio of the distance the protein
has moved from its’ point of origin (the beginning of the separating gel)
relative to the distance the tracking dye has moved (the gel front). The
ratio is abbreviated as Rf. Molecular weight is expressed in daltons.

Assignment
 Write the names of the standard proteins used..
 Get the molecular weight of the standard proteins.
 Convert the MW to log MW.
 Calculate the relative mobility of each std. protein:
Rf = distance migrated by the std. protein
distance migrated by the tracking dye
 Plot the Rf (x-axis) versus the log MW (y-axis). A straight line will be
obtained.
 Use the graph to obtain the log MW of the unknowns.
 Finally, convert the log MW to MW and construct a plot of the Rf versus Log
MW and fill in the following table.

Standard protein Standard Relative

name protein MW Log MW Distance (cm) mobility (Rf)
(Dalton)

Tracking dye

Unknown 1

Unknown 2 60

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Answer the following questions:
1. What will be the MW of the unknown proteins?
2. What will be the relative mobility of a protein having a MW of 90,000 Da?
3. What will be the distance traveled by the protein referred to in Q2?
4. A tri-meric protein having a molecular weight of 210 Da in gel filtration. The
SDS-PAGE resolved only two bands of 100 and 60 Da. How would you interpret
these data?
5. What are the major differences between the stacking and separating gels?
6. How can you change/control the pore size of the separating gel?
7. Describe the pattern of separation in a typical gel?
8. Study the following gels, which show the effect of the gel concentration (%T).
Briefly, describe this effect?

9. Complete the following table by briefly describing the function of each reagent or
chemical used in SDS-PAGE.

chemical Function or purpose

Acrylamide
Bisacrylamide
TEMED
Ammonium
persulfate
Sodium
Dodecyl
Sulfate
Bromophenol

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 blue
Coomassie
Brilliant Blue
(CBB)

List the references that you have used to find out this information
following this style:

References

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https://www.mayoclinic.org/diseases-conditions/heart-disease/in-
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Lindshield, B. (2018). Kansas State University Human Nutrition (FNDH

400) Flexbook. 1st edition. Manhattan, New York City: New Prairie
Press, pp. 103.

Smith, S. (2009). Mechanism of chain length determination in

biosynthesis of milk fatty acids. Journal of Mammary Gland Biology and
Neoplasia, 14(3), 245-260.

Samiee, K., Rustaiyan. A (2015). Omega-3 Fatty Acids Composition and

Lipid Content from Liver and Muscle Tissues of Scomberomorus
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Geleijnse, J. M., Giltay, E. J., Grobbee, D. E., Donders, A. R., Kok, F. J.

(2002). Blood pressure response to fish oil supplementation:
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