Molecular Techniques and Methods

SDS-Polyacrylamide Gel Electrophoresis

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

In SDS polyacrylamide gel electrophoresis, proteins are separated on the basis of their sizes, by the sieving effect of the polyacrylamide gel matrix. In this method, proteins are reacted with the anionic detergent SDS (sodium dodecylsulfate, or sodium lauryl sulfate) to form negatively charged complexes. The amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Per 1 g protein, about 1.4 g SDS is bound. The proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate elipsoid or rod of a length roughly proportionate to the protein's molecular weight. Thus, proteins of either acidic or basic pI form negatively charged complexes that can be separated on the bases of differences in charges and sizes by electrophoresis through a sieve-like matrix of polyacrylamide gel.
Stacking gel system employs the principles of isotachophoresis, which effectively concentrates samples from large volumes into very small zones, that then leads to better separation of the different species. The system is set up by making a Stacking Gel on top of the Separating Gel, which is of a different pH. The protein sample is introduced to the system at the Stacking Gel. With an electric field applied, ions move towards the electrodes, but at the pH prevailing in the Stacking Gel, the protein-SDS complexes have mobilities intermediate between the Cl- ions (present throughout the system) and glycinate ions (present in the reservoir buffer). The Cl- ions have the greatest mobility. The following larger ions concentrate into narrow zones in the Stacking Gel, but are not effectively separated there. When the moving zones reach the Separating Gel, their respective mobilities change in the pH prevailing there and the glycinate ion front overtakes the protein-SDS complex zones to leave them in a uniformly buffered electric field to separate from each other according to size and charge.

  • The gel system described in this procedure is suitable for electrophoresis of proteins in the molecular weight range of 10,000-100,000. Smaller proteins move at the front or form diffuse, fast-moving bands, whereas larger proteins hardly enter the gel, if at all.




  • MATERIALS AND SOLUTIONS

    30% Acrylamide:Bis Solution (37.5:1)
    Acrylamide ----------------------------------------------- 75 g
    Bis-acrylamide -------------------------------------------- 2 g
  • Add deionized H2O to make a final volume of ----------- 250 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    40% Acrylamide:Bis Solution (37.5:1)
    Acrylamide ----------------------------------------------- 100 g
    Bis-acrylamide -------------------------------------------- 2.7 g
  • Add deionized H2O to make a final volume of ----------- 250 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    4 x Separating Gel Buffer (200 ml)
    Tris (FW=121.1) ---------------------------------------- 36.3 g
    Deionized H2O ------------------------------------------ 150 ml
  • Adjust the pH to 8.8 with HCl.
    Add deionized H2O to make a final volume of ---------- 200 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    4 x Stacking Gel Buffer (50 ml)
    Tris (FW=121.1) ---------------------------------------- 15.1 g
    Deionized H2O ------------------------------------------ 40 ml
  • Adjust the pH to 6.8 with HCl.
  • Add deionized H2O to make a final volume of ---------- 50 ml
  • Store in a brown glass bottle for 3 months at 4oC.


    10% Ammonium Persulfate (10 ml)
    Ammonium persulfate ----------------------------------- 1.0 g
    Deionized H2O ----------------------------------------- 10 ml
  • Keep in a brown glass bottle at 4oC.


    Electrophoresis Buffer (2 liter)
    0.192 M Glycine --------------------------------------- 28.8 g
    0.025 M Tris (FW=121.1) ------------------------------- 6.0 g
    0.1% SDS ---------------------------------------------- 20 ml of 10% SDS
    Add deionized H2O to make a final volume of --------- 2 liter
    The solution should be at about
    pH 8.3 without adjustment.
    This solution is readily made fresh each time.


    H2O-Saturated n-Butanol (55 ml)
    n-Butanol --------------------------------------------- 50 ml
    Deionized H2O ----------------------------------------- 5 ml
    Combine in a bottle and shake. Use the top phase to overlay gels.
    Store at room temperature indefinitely.



    2 x Sample Buffer (10 ml)
    4 x Stacking Gel Buffer (pH 6.8) ---------------------- 2.5 ml
    4% SDS ------------------------------------------------ 4 ml of 10% SDS
    20% Glycerol ------------------------------------------ 2 ml of 100% Glycerol
    0.2% Bromophenol Blue --------------------------------- 400 ul of 5% Bromophenol Blue
    0.2 M DTT --------------------------------------------- 2 ml of 1 M DTT
    Store 0.5 ml aliquots at -20oC for 6 months.


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


    Destaining Solution (1 liter)
    Methanol ----------------------------------------------- 100 ml
    Glacial Acetic acid ------------------------------------ 100 ml
    Deionized H2O ------------------------------------------ 800 ml
    Mix thoroughly. Use when freshly made.




    PROCEDURES

    1. Thoroughly clean and dry the glass plates and three spacers, then assemble them with bulldog clips. Clamp the chamber in an upright, level position.

    2. Prepare 10 ml Separating Gel Mixture as follows.

    4%
    6%
    8%
    10%
    12%
    40% Acrylamide:Bis Solution (37.5:1)
    1 ml
    1.5 ml
    2 ml
    2.5 ml
    3 ml
    4 x Separating Gel Buffer
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    2.5 ml
    10% SDS
    0.1 ml
    0.1 ml
    0.1 ml
    0.1 ml
    0.1 ml
    50% Glycerol
    3.6 ml
    3.6 ml
    3.6 ml
    0 ml
    0 ml
    ddH2O
    2.8 ml
    2.3 ml
    1.8 ml
    4.9 ml
    4.4 ml
    Total volume
    10 ml
    10 ml
    10 ml
    10 ml
    10 ml
    10% Ammonium persulfate
    50 ul
    50 ul
    50 ul
    50 ul
    50 ul
    TEMED
    5 ul
    5 ul
    5 ul
    5 ul
    5 ul



    4. Mix gently and use
    immediately (because polymerization starts when the TEMED is added). Carefully pour the freshly mixed solution into the chamber without generating air bubbles. Pour to a level about 1 cm below where the bottom of the well-forming comb will come when it is in position.

    5. Carefully overlayer the acrylamide solution with H2O-saturated n-butanol without mixing to eliminate oxygen and generate a flat top to the gel.

    6. Polymerize the acrylamide for 1 hour.

    7. Prepare 5 ml, 4% Stacking Gel Solution as follows:

    40% Acrylamide:Bis Solution (37.5:1)
    0.5 ml
    4 x Stacking Gel Buffer
    1.25 ml
    10% SDS
    0.05 ml
    ddH2O
    3.2 ml
    10% APS
    25 ul
    TEMED
    2.5 ul



    9. Mix gently and use
    immediately.
    Pour off the n-butanol from the polymerized Separating Gel, wash the gel top with water, and fill the gap remaining in the chamber with the Stacking Gel mixture. Insert the comb.

    10. Polymerize the acrylamide for 1 hour.

    11. When the Stacking Gel has polymerized, remove the comb without distorting the shapes of the well. Remove the clips holding the plates together, and install the gel in the apparatus.

    12. Fill apparatus with Reservoir Buffer. Push out the bottom spacer from the gel and remove bubbles from both the top and underneath of the gel. Use the gel immediately.

    13. While the gel is polymerizing, prepare samples for electrophoresis.
    Dissolve the protein sample solution in a same volume of 2 x Sample Buffer, or dissolve a dry sample in 1 x Sample Buffer. The concentration of sample in the solution should be such as to give a sufficient amount of protein in a volume not greater than the size of the sample well.

    14. Heat sample solutions in boiling water for 2 minutes.
    (Some proteins may react adequately with SDS within a few minutes at room temperature.)
    (The bromophenol blue dye in Sample Buffer indicates when the sample solution is acidic by turning yellow. If this happens, add a little NaOH, enough to just turn the color
    blue.)

    15. Load the gel with 10-30 ul Protein Sample Solution by pipet.

    16. Start electrophoresis immediately by turning on power. On a gel of 1 mm thickness and 15 cm length, an applied voltage of about 150 volts gives a current of about 20 mA (falling during electrophoresis if constant voltage is employed). The bromophenol blue dye front takes about 3 hours to reach the bottom of the gel. Greater voltage speeds up electrophoresis, but generates more heat in the gel.
  • For Bio-Rad Protean II xi gel (20 x 16 cm x 1.5 mm), total volt.hour = 1,200 - 1,300 V.H.

    17. Remove the gel from between the glass plates.

    18. Stain the gel in the Staining Solution for 2-3 hours.

    19. Remove the dye that is not bound to protein in Destaining Solution. After about 24 hours, with gentle agitation and several changes of Destaining Solution, the gel background becomes colorless and leaves protein bands colored blue, purple, or red. Coomassie Brilliant Blue R250 and PAGE Blue 83 each visibly stain as little as 0.1-1 ug of protein in a band of about 1 cm width.




    NOTES

  • The reducing agent (DTT) in the 2 x Sample Buffer reduces intermolecular disulphide bridges and so destroys quarternary structure and separates subunits, and also oxidizes intramolecular disulfide bonds to, ensure maximal reaction with SDS.


  • The glycerol in the 2 x Sample Buffer increases the density of the sample, to aid the loading of it onto the gel.


  • The bromophenol blue dye in the 2 x Sample Buffer aids loading of the sample, by making it visible, and indicates the position of the front of electrophoresis in the gel. The bromophenol blue also indicates when the sample solution is acidic by turning yellow. If this happens, add a little NaOH, enough to just turn the color blue.


  • The degassing stage removes oxygen, which inhibits polymerization by virtue of mopping up free radicals, and also discourages bubble formation when pouring the gel.


  • The polymerization of acrylamide and bisacrylamide is initiated by the addition of TEMED and ammonium persulfate. The ammonium persulfate activates the TEMED and leaves it with an unpaired electron. This radical reacts with an acrylamide monomer to produce a new radical that reacts with another monomer, and so on to build up a polymer. The bis acrylamide is incorporated into polymer chains this way and so forms crosslinks between them.





  • KIT INFORMATION




    REFERENCES

  • Maguire GF, Lee M, Connelly PW (1989) Journal of Lipid Research. 30, 757-761.

  • Deyl, Z., 1979, Electrophoresis. A survey of techniques and applications. Part A: Techniques. Elesevier, Amsterdam.


  • Laemmli, U.K., 1970, Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.


  • Studier, F.W., 1973, Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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