SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis  (SDS-PAGE) was introduced in  1967 by Shapiro and has since become one of the most popular PAGE methods.  The  method is dependent in the first instance upon the interaction of the  protein  molecule with SDS. This is a detergent having a 12 carbon hydrophobic tail and a hydrophilic, sulfonic acid head group (I).

Proteins are denatured by boiling in the presence of SDS.  The SDS molecules interact  with the  proteins  to  give rod-like complexes, containing a constant ratio of ca. 1.4 mg of SDS per mg of protein. At this level the negative charge on the SDS is sufficient to  mask the  charge on the  protein  and all proteins  consequently  have  essentially  the  same charge/mass ratio and an anodic migration.

The lack of charge differences between different protein/SDS complexes means that the stacking phase will not  be as effective  as in conventional disc electrophoresis.  However, Laemmli has devised a convenient method whereby disc PAGE and  SDS-PAGE can be conducted with the same set of reagents, simply with or without SDS.  The  Laemmli method has become one  of the  most  popular  PAGE methods  in use today. However, the Laemmli method does not separate small proteins very well and an alternative SDS-PAGE method,  using  Tricine  buffer,  has been described by Shagger and Von Jagow. The Tricine method gives uniquely good separation of proteins under 20 kDa. As the intrinsic  charge differences  between proteins  is masked by the SDS, separation of proteins is due solely to  differences in size and hence the method can be used to determine molecular sizes.  A linear relationship between mobility and log MW obtains over a molecular weight range dependent upon  the  gel pore  size.  The  gel  can  thus  be standardized with proteins  of known  molecular  weight  and  subsequently used to  estimate  the  molecular weights of unknowns.  Mobility  is conveniently expressed as relative mobility (R_m), i.e. mobility relative to the bromophenol blue tracker dye.

Figure 1.  Standard curve for estimation of protein MWs by  SDS-PAGE.

Some caveats apply to the estimation  of molecular weights.  If proteins are not reduced, then disulfide bridges may constrain the structure and prevent formation  of the  rod-like complexes.  This will result in an incorrect apparent MW.  On the  other  hand, this provides a way of detecting the existence  of disulfide bridges.  The  glyco  moiety  of glycoproteins will also not bind SDS, and yet  will contribute  to  the  steric resistance of the molecule.  In consequence, the molecular weights of glycoproteins tend to be overestimated.  Finally, boiling in  SDS not  only denatures the protein  but will dissociate the  subunits of oligomeric proteins. The MWs  obtained  by  SDS-PAGE will therefore  be  of  the subunits and not of the intact protein.

An SDS-PAGE zymogram for proteinases

Zymography is a method for the detection of a specific enzyme among the bands separated by electrophoresis.  The  method  usually relies on an enzyme specific reaction to  generate  a colour to  reveal the position of the enzyme.  Usually zymogram methods cannot be applied to SDS-PAGE as proteins  are  normally  denatured  in  this  technique.

However, Heussen and Dowdle have devised an ingenious  SDS-PAGE zymogram method for proteinases.  The method  depends upon combining the  proteinase-containing mixture with SDS, but without boiling the solution.  In  this  form  the  SDS  can  apparently  combine  only partially with the protein, perhaps “tweaking” its conformation slightly and suppressing the activity of proteinases.

The SDS/protein mixture is separated by electrophoresis in a polyacrylamide gel containing a low concentration  of gelatin (less than 1%). The proteinase/SDS complex does not bind to the gelatin, as would a free  proteinase,  and migrates  as a narrow  band.  Subsequent incubation in a non-ionic detergent, such as Triton  X-100, removes the  SDS from the proteinase and reconstitutes  its activity.  The  reactivated  proteinase digests away part  of the  gelatin and its position  can be detected by subsequently staining the gel.  The proteinase-digested gelatin appears as a clear band on the blue stained gel.

References 

Dennison, C. (2002). A guide to protein isolation . School of Molecular mid Cellular Biosciences, University of Natal . Kluwer Academic Publishers new york, Boston, Dordrecht, London, Moscow .

Shapiro, A. L., ViÒuela, E. and Maizel, J. V. Jnr (1967) Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.  Biochem. Biophys. Res. Commun. 28, 815-820.

Weber, K.  and Osborn, M.  (1969) The reliability  of molecular weight determinations by dodecyl-sulphate-polyacrylamide gel electrophoresis. J.  Biol.  Chem.  244, 4406-4412.

Shgger, H.  and von Jagow, G.  (1 987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from  1-100 kDa. Anal. Biochem. 166, 368-379.

Heussen,  C.  and Dowdle, E.  B.  (1996) Electrophoretic  analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem.  102, 196-202.

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