Molecular Techniques and Methods

Acetic Acid-Urea Polyacrylamide Gel Electrophoresis

Copy Right © 2001/ Institute of Molecular Development LLC


In an Acetic Acid-Urea polyacrylamide gel electrophoresis, both the molecular size and charge act as bases for protein separation. The pH in a system is commonly about 3.0. Since the pKa values of the side chain carboxyl groups of glutamic acid and aspartic acid are about 4.2 and 3.8, respectively, even these amino acids will contribute little to the negative charge on a protein at pH 3.0. Thus at pH 3.0, all proteins are likely to be positively charged (+) and to travel towards the cathode (-) in an electric field.
In such an Acetic acid-Urea polyacrylamide gel electrophoresis system, two proteins of similar size but different charge may be separated from each other. Since SDS gels may be unable to achieve this end, these two electrophoresis systems usefully complement each other for analysis of small amounts of proteins. Proteins that might be studied in the Acetic acid-Urea polyacrylamide gel electrophoresis system are minor primary structure variants of slightly different charge, or modified forms of the same protein. A protein that has had some threonine or serine side chains phosphorylated, or lysine side chains acetylated, will be more acidic than the unmodified form of the same protein, and so will have a different electrophoretic mobility in the Acetic acid-Urea polyacrylamide gel electrophoresis system. Urea, the hydrogen bond-breaking agent, is added to the Acetic acid-Urea polyacrylamide gel electrophoresis system in amounts traversing its entire range of solubility. Urea increases the frictional coefficient of proteins and so alters their electrophoretic mobilities.


30% Acrylamide:Bis Solution (67:1) (250 ml)
Acrylamide --------------------------------------------- 73.8 g
Bis acrylamide ------------------------------------------ 1.1 g
Add Deionized H2O to make a final volume of -------- 250 ml
Filter the solution by 0.4 um millipore membrane.
Keep in brown glass bottle at 4oC.

10% Ammonium Persulfate (10 ml)
Ammonium persulfate ----------------------------------- 1 g
Add Deionized H2O to make a final volume of --------- 10 ml
Keep in brown glass bottle at 4oC.

Electrophoresis Buffer (pH 3.0) (1 liter)
0.9M Glacial Acetic acid ------------------------------- 51.5 ml of 17.5 M Glacial Acetic acid
Deionized H2O ---------------------------------------- 948.5 ml

Denaturing Sample Buffer (10 ml)
1 M HCl ----------------------------------------------- 1 ml of 10 M HCl
2-mercaptoethanol ------------------------------------- 0.5 ml
Urea --------------------------------------------------- 5.4 g
Pyronin Y (0.4% w/v solution in water) ----------------- 0.5 ml
Add Distilled H2O to make a final volume of ----------10 ml
Store at -20oC for several weeks.

Native Sample Buffer (10 ml)
0.9 M Glacial Acetic acid ------------------------------- 0.52 ml of 17.5 M Glacial Acetic acid
30% Sucrose ------------------------------------------- 3 g
Pyronin Y (0.4% w/v solution in water) ----------------- 0.5 ml
Add Distilled H2O to make a final volume of -----------10 ml
Store at -20oC for several weeks.

Staining Solution (250 ml)
Coomassie Brilliant Blue R250 -------------------------- 0.25 g
(or, PAGE Blue 83 ------------------------------------- 0.25 g)
Methanol ---------------------------------------------- 125 ml
1.75 M Glacial Acetic acid ----------------------------- 25 ml of 17.5 M Glacial Acetic acid
Deionized H2O ----------------------------------------- 100 ml
Dissolve the dye in the methanol, then add the acid and water. If dissolved up in a different order the dye's staining behavior may differ.

Destaining Solution (1 liter)
Methanol ----------------------------------------------- 100 ml
1.75 M Glacial Acetic acid ------------------------------ 100 ml of 17.5 M Glacial Acetic acid
Deionized H2O ------------------------------------------ 800 ml
Mix thoroughly. Make when required and use fresh.


1. Assemble clean glass plates and spacers into the form of a chamber. Clamp it in an upright, level position.

2. Prepare the Separating Gel as follows.

30% Acrylamide:Bis Solution (67:1)
20 ml
2.5 M Urea
4.5 g
Glacial Acetic acid
1.57 ml
TEMED (N,N,N',N'-tetramethylethylenediamine)
100 ul
Add Deionized H2O to make a final volume of
30 ml

  • Degas the solution.

    Add 10% Ammonium Persulfate ----------------------------- 0.55 ml

    3. Mix gently and pour immediately into the chamber and remove any air bubbles present.
    (Pour to a level about 0.5 cm below where the bottom of the well-forming comb will come when it is in position.)

    4. Carefully overlay the acrylamide solution with 2-butanol, without mixing.
    (This insulates the solution from oxygen and generates a flat top to the gel.)

    5. Polymerize the acrylamide for 1 hour at room temperature.

    6. Prepare the Acidic Stacking Gel as follows.

    30% Acrylamide:Bis Solution (67:1)
    1.25 ml
    2.5 M Urea
    0.75 g
    Glacial Acetic acid
    78.2 ul
    TEMED (N,N,N',N'-tetramethylethylenediamine)
    25 ul
    Add Deionized H2O to make a final volume of
    5 ml

  • Degas the solution.

    Add 10% Ammonium Persulfate ----------------------------- 0.24 ml

    7. Pour off the 2-butanol from the polymerized Separating Gel, wash the gel top with deionized H2O, and then fill the gap remaining in the chamber with the Acidic Stacking Gel mixture.

    8. Insert the well-forming comb and allow to polymerize for 1 hour.

    9. When the upper gel has set, remove the comb without breaking or distorting the sample wells.

    10. Install the gel in the apparatus and fill the reservoirs with Electrophoresis Buffer. Push out the bottom spacer from the chamber and, with the cathode (-) at the bottom end of the gel.

    11. Pre-run the gel at 180 volts and room temperature until the current falls to a steady level.
    (This process may take about 3-5 hours, but may be done conveniently overnight. The pre-electrophoresed gel may be stored under fresh Electrophoresis Buffer for several days at room temperature.)

    12. Prepare samples in small volume (10-30 ul) of Denaturing Sample Buffer (or, in Native Sample Buffer for separation of nondenatured proteins) for electrophoresis. Dry samples will be dissolved directly in Denaturing Sample Buffer. The concentration of protein in the solution should be as great as possible so that the volume (10-30 ul) of solution loaded onto the gel is as small as possible.

    13. Prepare samples to load the gel. Use fresh Electrophoresis Buffer in the apparatus. Take up the required volume (10-30 ul) of sample solution in a pipet and carefully inject it into a sample well, loading it through the reservoir buffer without mixing.

    14. Electrophoresis at 180 volts for 4-7 hours.
    (Decreasing the voltage will prolong the run, whereas an increased voltage will generate more heat, which may distort the appearance of the protein bands.)

    15. At the end of electrophoresis, remove the gel from the glass plates and immerse it in 500 ml Staining Solution for several hours to overnight with gentle agitation.

    16. Rinse the unbound-dye by washing the gel in several changes of Destaining Solution. After destaining, protein bands are seen to be colored blue to purplish-red.
    (Coomassie Brilliant Blue R250 and PAGE Blue 83 each visibly stain as little as 0.1-1 ug of protein in a 1 cm wide band.)


  • The purpose of the Acidic Stacking Gel is to provide a medium in which sample wells can be formed and from which the well-forming comb can be readily removed (20%T gel tends to break when the comb is removed). Since the Acidic Stacking Gel contains weaker acid than does the Separating Gel, its pH is slightly higher. However, the purpose of this design is that the Acidic Stacking Gel has lower conductivity than the rest of the system and when the electric field is applied this has a small band-sharpening effect.

  • Addition of TEMED and ammonium persulfate to the gel mixture initiates its polymerization. This occurs by their interaction and formation of a TEMED radical that reacts with an acrylamide monomer. This in turn produces a radical that reacts with another acrylamide monomer.

  • The 9 M urea in the Sample Buffer disrupts aggregates, and also increases the density of the solution (which aids in the loading of the sample beneath the less dense Electrohporesis Buffer).

  • The 2-mercaptoethanol reduces intermolecular and intramolecular disulfide bonds and destroys tertiary or quaternary protein structures.

  • The pre-electrophoresis treatment of the gel before addition of the samples removes ammonium persulfate and other ions that would otherwise slow up the rate of sample electrophoresis and spoil the resolution of protein bands.

  • The Acetic acid-Urea system described may be adapted for separation of nondenatured proteins, which can be detected in the gel by their enzymatic properties and undenatured from the gel. For this purpose, samples are dissolved in the Native Sample Buffer.



  • Gordon, A.H., 1969, Electrophoresis of proteins in polyacrylamide and starch gels, In "Laboratory Techniques in Biochemistry and Molecular Biology", Vol. 1 (eds. Work, T.S., and Work, E.), pp.1-149. North Holland, Amsterdam, London.

  • Hardison, R., and Chalkley, R., 1978, Polyacrylamide gel electrophoretic fractionation of histones. In Methods in Cell Biology, Vol. 17 (eds. Stein, G., Stein, J., and Kleinsmith, L.J). Academic Press, New York.

  • Hrkal, Z., 1979, Gel-type techniques. In Electrophoresis. A survey of techniques and applications (ed. Deyl, Z.). Elsevier, Amsterdam.

  • Kistler, W.S., Geroch, M.E., and Williams-Ashman, H.G., 1973, Specific basic proteins from mammalian testes. Isolation and properties of small basic proteins from rat testes and epididymal spermatozoa. J. Biol. Chem. 248, 4532-4543.

  • Panyim, S., and Chalkley, R., 1969, High resolution acrylamide gel electrophoresis of histones. Arch. Biochem. Biophys. 130, 337-346.

  • Spiker, S., 1980, A modification of the acetic acid-urea system for use in microslab polyacrylamide gel electrophoresis. Anal. Biochem. 108, 263-265.

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