SDS electrophoresis
SDS electrophoresis SDS being the abbreviation for sodium dodecyl sulphate which separates exclusively according to molecular weight. By loading with the anionic detergent SDS, the charge of the proteins is so well masked that anionic micelles with a constant net charge per mass unit result: 1.4 g SDS per g protein. In addition, the differences in molecular form are compensated by the loss of the tertiary and secondary structures because of the disruption of the hydrogen bonds and unfolding of the molecules.
Exponential gradients areformed when the mixing chamber is sealed. The volume in the mixing chamber stays constant, the same quantity of dilute solutionflows in as solution out of the mixing chamber.
Disulfide bonds which can form between cysteine residues can only be cleaved by a reducing thiol agent such as 2mercaptoethanol or dithiothreitol. The SH groups are often protected by a subsequent alkylation with iodoacetamide, iodoacetic acid or vinylpyridine.
The unfolded amino acid chains, bound to SDS form ellipsoids with identical central axes. During electrophoresis in restrictive polyacrylamide gels containing 0.1% SDS there is a linear relationship between the logarithm of the molecular weight and the relative distance of migration of the SDSpolypeptide micelle.
Gels with a pore gradient have a wider separation interval and a larger linear separation interval than gels with a constant pore size. In addition, sharper bands result since a gradient gel minimizes diffusion. The molecular weight of the proteins can be estimated with a calibration curve using marker proteins.
This linear relationship is only valid for a certain interval which is determined by the ratio of the molecular size to the pore diameter.
For separation of physiological fluids or analysis of urine proteins for example, the reduction stop is left out to prevent the breakdown of the immunoglobulins into subentities. In these cases the incomplete unfolding of certain proteins must be taken into account and therefore the molecular weight cannot be determined exactly.
For example when it is not reduced, albumin shows a molecular weight of 54 kDa instead of 68 kDa since the polypeptide chain is only partially unfolded.
There are a number of practical advantages to SDS electrophoresis:
After electrophoretic transfer on an immobilizing membrane, the SDS can be removed from the proteins without eluting the proteins themselves.
SDS electrophoresis can be carried out in a continuous phosphate buffer system or in a discontinuous system:sharp zones no strong acids are necessary
Silver staining increases the limit of detection tenfold
Laemmli has directly taken over the disc electrophoresis method according to Ornstein and Davis, for proteins charged with SDS, though the jumps in pH value and ionic strength are not necessary.
· Because the proteinSDS micelles have a very high negative charge, the mobility of glycine which is very hydrophilic and thus does not bind to SDS, is lower than that of the proteins in the stacking gel at the beginning of electrophoresis, even at pH 8.8.
· During stacking no field strength gradient results, since there are no charge differences within the sample: so no low ionic strength is necessary.
This means that SDS disc electrophoresis gels can be cast in one step: Glycerol is added to the resolving gel and then the stacking gel, which contains the same buffer but no glycerol, is directly cast over it. In addition, the run time is shorter since it starts more quickly.
Since there are no diffusion problems between the stacking and the resolving gel buffers with these gels, they can be stored longer than conventional disc gels. Yet their storage capacity is limited by the high pH value of the gel buffer, since, after about 10 days, the polyacrylamide matrix starts to hydrolyze.
After empirical assays, a Trisacetate buffer with a pH of 6.7 has proven to have the best storage stability and separation capacity. Tricine is used instead of glycine as the terminating ion.
SDSelectrophoresis of low molecular weight peptides: the resolution of peptides below 14 kDa is not sufficient in conventional TrisglycineHCI systems. This problem has been solved by the development of a new gel and buffer system by Schägger and Jagow. In this method an additional spacer gel is introduced, the molarity of the buffer is increased and tricine used as terminating ion instead of glycine. This method yields a linear resolution from 100 to 1 kDa.
Glycoproteins: glycoproteins migrate too slowly in SDS electrophoresis, since the sugar moiety does not bind to SDS. When a TrisborateEDTA buffer is used, the negative sugar moieties are also negatively charged so the speed of migration increases.
Glycine and SDS are not needed for anodic electrophoresis buffers which reduces the costs. The discontinuity of the anions is very important and the various gel porosities are very useful.
The overlayering of the resolving gel with butanol for example can thus be avoided.
For readymade gels with higher storage capacity, another buffer system with pH values around 7 should be chosen.
Since Tricine is much more expensive than glycine, it is only used at the cathode, the anode contains Trisacetate.
Cationic detergents: strongly acidic proteins do not bind to SDS and very basic nucleoproteins behave abnormally in SDS gels. The alternative is to use a cationic detergent, cetyltrimethylammonium bromide (CTAB) in an acidic medium at pH 3 to 5 is recommended. This also allows a separation according to the molecular weight in the direction of the cathode. This cationic detergent also causes less damage to the protein than SDS, so CTAB electrophoresis can be used as a form of native electrophoresis.
SDS electrophoresis in washed, dried and rehydrated polyacrylamide gels: the TrisHCI /Trisglycine buffer system shows very poor results. However good results are obtained with the Trisacetate / Tristricine system. In this method, the gel is rehydrated in Trisacetate pH 8.0 using a horizontal tray. If, for highly concentrated Protein samples, a discontinuity in pH and molarity between stacking and resolving gel is required, the stacking zone can be selectively equilibrated in a higher diluted Trisacetate buffer pH 5.6 using a vertical chamber.
Native electrophoresis in amphoteric buffers: the polymerization catalysts can be washed out of the polyacrylamide gels on support films used in horizontal systems with deionized water. By equilibration with amphoteric buffers such as HEPES, MES or MOPS for example, there is a wide spectrum for electrophoresis under native conditions.
The performance of SDS buffer systems are obviously highly influenced by catalysts and / or monomers of acrylamide.
This procedure of washing, drying, rehydration and equilibration con could be performed with gels polymerized on carrier films, which are used in horizontal gels
The ionic catalysts APS and TEMED would destabilize these buffer system