Gel based proteomics

2D PAGE = 2D gel already almost been alternative by LC-MS
蛋白质的指纹： 电荷性质（等电点） 分子量大小（kDa） 疏水性（亲水性） 亲和性质（抗原抗体）

1. What is 2D PAGE:
   a kind of electrophoresis, which is a common method for molecules to be separated in a particular medium(liquid or gels), by means of an electric field. Depending on conditions used this separation is based on charge, mass/charge, mass. e.g. used in DNA/protein electrophoresis.

2. How does 2D PAGE work?
   Protein separated based on extraction a sample from tissue, then protein separated based on pI (Isoelectric point 等电点) value, happened on a small strip(depend on pH). And this strip is then used to prepare for 2nd dimension SDS-PAGE.

• 1st dimension iso electrofocusing（第一向） :
  根据蛋白质的等电点不同， 在变形条件下等电聚焦分离蛋白。

Proteins are separated on a polyacrylamide gel strip on which a pH gradient is present. During electrophoresis, proteins move to pH=pI. At this point, electrophoretic mobility is 0 (net charge of the protein is 0).
蛋白质是两性电解质，在特定的pH溶液中所带正电荷数恰好等于负电荷数。此时蛋白在电场中不再移动，此溶液的pH称该蛋白质的等电点。

• Originally: (mobilizable)
  Carrier ampholytes 载体两性电解质: compounds that have both negative and positive charges, and spontaneously create a pH gradient when an electric field is applied.

• Today : Immobilized pH gradients (commercial)

  • 2nd dimension: SDS-PAGE（第二向）
    根据蛋白质的分子量不同，在sds电泳中分离蛋白
    IEF strip is loaded in SDS, proteins are separated by size.

3. 2D PAGE result: a gel containing spots representing the proteins in the sample.
   ◦ Extreme pI value are difficult to approach,
   ◦ Very hydrophobic proteins more stringent conditions(严格条件)
   ◦ Classical SDS_PAGE poorly resolves protein <10 kDA. -> This can be improved by using a different buffer system

4. Protein detection methods in 2D PAGE:
   classical : commassie blue, and silver staining
   recently: fluorescent dyes . Good detection limit
   advantage: accurate

5. 2D- PAGE for expression profiling

   • create protein maps
   • protein list: typically detect some 500 most abundant proteins

6. 2D-PAGE application: image analysis, match the protein in different gels
   avoid the image matching : DIGE

7. Example: from the plant field

8. Conclusion 2D-gel
   ad: widely used, affordable, top down technique
   dis: require servere efforts in sample preparation, image analysis is a heavy task.
   poor dynamic range, typically for visualizing housekeep proteins unless specific enrichment techniques
   not good reproducibility, >4 biological replicates, poor lab to lab reproduction.

9. How can we identify proteins from a gel ?
   early days: Edman sequencing
   western blotting

   MASS Spectrometry