Denaturing gel electrophoresis

In comparing to regular gel electrophoresis, denaturing gel electrophoresis requires special chemical reagents in gel, running buffer, and loading buffer. These chemicals denatures sample molecules so that they migrate in rates corresponding to their size only. There are two types of denaturing gel electrophoresis, denaturing agarose gel electrophoresis for RNA samples and denaturing PAGE gel electrophoresis for protein samples. 

The difference between agarose denaturing gel electrophoresis and PAGE denaturing gel electrophoresis are shown below:

1. Difference in gel systems : 

Horizontal submarine gel equipment is used for agarose denaturing gel electrophoresis of RNA samples.

Vertical slab gel system is used for PAGE denaturing gel electrophoresis of proteins.

2. Difference in denaturing chemicals:

MOPS and formamide are used for RNA denaturing gel electrophoresis.

SDS is used for Protein denaturing gel electrophoresis.

Urea is used for DNA denaturing gel electrophoresis.

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MOPS denaturing gel electrophoresis

  10 x MOPS denaturing buffer:

0.2M MOPS (morpholinopropanesulphonic acid)
50mM sodium acetate
5mM EDTA

RNA denaturing buffer:

10ml 100% deionized formamide
3.5ml 40% formaldehyde
1.5ml 10 x MOPS buffer
Formamide should be deionized by stirring 100ml with approximately 20g of Amberlite MB3 (or MB1) ion exchange resin before application to denaturing agarose gel.

Denaturing agarose gel is prepared by melting the required amount of agarose in distilled water, cooling to approximately 60°C (hand hot) and adding 40% formaldehyde and 10 x MOPS to give 2.2M formaldehyde and 1 x MOPS, respectively. 

Example: For 50ml of a 1% agarose gel, melt 0.5g agarose in 37ml H2O, cool to hand hot, add 5ml 10 x MOPS buffer and 8.75ml 40% formaldehyde.

denaturing agarose gel electrophoresis is in 1 x MOPS, 2.2M formaldehyde.

RNA samples are prepared by adding up to 25mg of RNA in a maximum of 5ul sterile H2O, to 15ul RNA denaturing buffer. 1ul 10mg / ml ethidium bromide is added to aid visualization of RNA after denaturing agarose gel electrophoresis.

Immediately prior to loading, RNA samples should be heated to 65°C for 10 minutes to denature any secondary structure, cooled on ice for 2 minutes and then 2ul of denaturing agarose gel loading buffer is added.

Samples are loaded onto the denaturing agarose gel and electrophoresis using Biokeystone high performance gel systems.

SDS-PAGE of Proteins

Separation of proteins under denaturing conditions in SDS denaturing gel electrophoresis

Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.