SDS-PAGE and Native PAGE (due November 25th, 2019)
In SDS-PAGE complexes are separated to their subunits, proteins are denatured and covered by SDS molecules at a
ratio of approximately 1 SDS molecule per 2 amino acids. Thus any charge that the protein might have is masked by
he huge negative charge by the SDS molecules and migration and thus separation of proteins depends mainly on
their size.
That's why SDS page is commonly used for determing approximate molecular weight of proteins, for following the
progress of protein purification, etc.
In native PAGE proteins retain their natural fold and can remain in complex. So the migration depends on the charge
of the protein, the size, shape and if it is in complex with other molecules or if it oligomerizes.
For a example a protein that forms tetramers will give one band in an SDS-PAGE that corresponds to the monomer
(provided that denaturation is complete) while on a native PAGE it can give more than one band, depending on the
amount of each species (monomer, dimer, trimer, tetramer)
From native PAGE usually in combination with other techniques you can see the oligomerization state of your
protein or study complexation reactions like protein-DNA (band-shift assays)
In Native PAGE,no denaturing agents are used to denature the protein sample.same buffers are used in all the three:
> the buffer,
> the gel and
>in the sample.
A single gel system is used to seperate the proteins based on their molecular weight. Using this technique,enzymes
can be seperated without denaturing them as they lose their activity upon denaturation.
In SDS-PAGE, an anionic detergent,SDS is used to, denature the protein sample and this sample is subjected to
electrophoresis.
two different gel types are used: stacking gel with pH-6.8 and a seperating gel of pH-8.8.
The buffer also varies with the gel and the tank.
The proteins get stacked between the glycine ions of tank buffer and chloride ions of buffer used to dissolve the gel
in the stacking gel. In separating gel,the glycine and chloride ions move faster, and the proteins form bands
according to their molecular weight. This is the most widely used technique for the separation of proteins and we
can determine their molecular weight by comparing the bands with the marker protein sample run aside.