2D electrophoresis

2-DE was first independently introduced by O'Farrell and Klose in 1975.
*Principle--:
 2D-PAGE is the primary technique for proteomics work. It separates the complex mixture of samples using two different properties of the proteins. In the first dimension, proteins are separated by the pI value and in the second dimension by the relative molecular weight.
Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels.
It is the method available which is capable of simultaneously separating thousands of proteins

This technique separate proteins in two steps , according to two independent properties :-
-First dimension is isoelectric focusing (IEF) which separates proteins according to their isoelectric point   
-secound dimension is SDS-polyacrylamide gel electrophoresis (SDS-page), which separates proteins according to their molecular weight (MW).

*Protocol :-

Isoelectric focusing (first dimension) 
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To prepare a thin layer IEF gel . Carrier ampholytes covering a suitable PH range
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Riboflavin are mixed with the acrylamide solution and the mixture is the poured over a glass plate which contains spacer
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The second glass plate is then placed on top of the first to form the gel cassette
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The gel polymerised by photopolymerisation  by placing the gel polymerised by photopolymerisation by place the gel in front of a bright light 
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After  2-3 hours , once the gel has set the glass plates are prised apart to reveal the gel stuck to one of the glass sheet 
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Electrode wicks , which are thick (3mm) and long strips of wetted filter paper ,a potential difference applied on it
(anode is phosphoric acid and cathod is sodium hydroxide)
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The power is then turned off and samples applied by laying on the gel ,small square of filtre paper soaked in the sample 
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A voltage is again applied for about 30 minutes to allow the sample for electrophoresis into gel at which time the paper square can be removed from the gel 
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To achieve rapid separation (2-3 hrs ) relatively high voltages (up to 2500 v) are used 
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As considerable heat is produced , gels are run on cooling plates (10°c) and power pack used to minimise thermal fluctuations 
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Following electrophoresis , the gel must be stained to detect the proteins 
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The gel is therefore first washed with fixing solution(ex. 10% trichloroacetic acid )
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The gel is again stained with coomassive Brilliant blue and then be stained
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Since , the proteins are in their native form enzymes can be detected in the gel either by washing the unfixed and unstained gel in an appropriate substrate or overlayering with agarose containing the substrate 

* SDS -Page (second dimension)-:

The first dimension (IEF) is carried out in polyacrylamide gels in narrow tubes (1-2mm internal diameter)
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The gel is then separated from the tube by applying slight pressure to one end 
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Incubated for 15 minutes in buffer containing SDS , then placed along the stacking gel of a SDS-added gel 
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Fixed in place by pouring molten agarose in electrophoresis buffer , over gel 
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Once the agarose has set , electrophoresis is start and the SDS bound proteins run into the gel , stack and separate according to size

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