2D PAGE (Polyacrylamide gel

electrophoresis)
Isoelectric focusing
1st dimension: Isoelectric focusing
Buffers for isoelectric focusing
• Rehydration buffers consists of ampholytes, CHAPS, Urea and Tracking
dye
• Equilibration buffer number 1: DDT (Reducing agent)- Breaks
disulphide bridges
• Equilibration buffer number 2: Iodoacetamide- adds carbamido
methyl groups to free cysteines
Sample loading onto the strip
• There are 2 ways to actually load the sample–you either include it in
the rehydration buffer or you load it in a small plastic cup at the end
of strip

• The strips are then placed in the IEF apparatus, filter paper wicks are
placed over the ends of the gel (these collect salts and
proteins/peptides that are outside of the pI range of the IEF strip).
Potential difference applied for ampholytes to create PH gradient
2nd dimension
• Direct staining is not possible, because ampholytes will stain as well. And it will be
completely blue. Hence fixing solution is used. Tricholoroactetic acid will precipitate
the proteins and much less ampholytes to be washed out.
• Isoelectric focusing takes 10-20 hours
• After IEF, strip is soaked in buffer containing SDS for 20 minutes
IPG Strip manufacturing
Tube gel

• Paper is cited 19,000 times

• Tube gels were difficult to handle, so in 1980
mobilized PH gradient strips were developed on
plastic packing
 Poured between glass plates

Occurs under bright light by decomposition of riboflavin

in to free radicals which initiate polymerization
Advantages of 2D Gel
See the practical procedure here
• https://www.youtube.com/watch?v=7R_R6mbqvFk&list=LLn19ykWo
pAtejN2mePS2gHw&index=14&t=0s