chromatofocusing, 2 de, ief

@ dimensional gel electrophoresis and chromatofocussing

1. Analytical and Biochemical Techniques

2. Chromatofocusing  is a protein-separation technique that allows resolution of single and other  ampholytes from a complex mixture according to differences in their  isoelectric point. Chromatofocusing utilizes ion exchange resins and is typically performed on fast protein liquid chromatography (FPLC) or similar equipment capable of producing continuous buffer gradients though this is not a requirement. In contrast to typical ion exchange chromatography , where bound molecules are eluted from the resin by increasing the ionic strength of the buffer environment, chromatofocusing e lutes bound species by altering the pH of the buffer. This changes the net surface charge of bound molecules, altering their affinity for the resin. As the changing pH of the buffer system traverses the pI of a given molecule, that molecule will elute from the resin as it will no longer possess a net surface charge (a requisite for molecular binding to ion exchange resins). Chromatofocusing

3. Chromatofocusing is a powerful purification technique with respect to proteins as it can resolve very similar species only differing by 0.02 pH units that may not separate well, or at all, using traditional ion exchange strategies. A major drawback to this technique is that some proteins will aggregate when present at relatively high concentrations and carry no net surface charge. This can cause blockage of the resin, which is highly problematic when using sealed columns of ion exchange resin on FPLC equipment, resulting in pressure build up and possible equipment failure. Apparent aggregation issues can sometimes be overcome by limiting the sample concentration and use of buffer additives that prevent aggregate formation.

4. ISOELECTRIC FOCUSING Electrophoretic method that separates proteins according to the iso -electric points Is ideal for seperation of amphoteric substances Seperation is achieved by applying a potential difference across a gel that contain a pH gradient Isoelectric focusing requires solid support such as agarose gel and polyacrylamide gel

5. Isoelectric focusing gels contain synthetic buffers called ampholytes that smooth the pH gradients. Ampholytes are complex mixtures of synthetic polyamino-polycarboxylic acids Commercially available ampholytes are- BIO-LYTE PHARMALYTE

6. It gives good separation with a high resolution compared to any other method Resolution depends on The pH gradient, The thickness of the gel Time of electrophoresis, The applied voltage, Diffusion of the protein into the gel .

7. At pH = pI , a protein will have no net charge  stop moving At any other pH in the gradient, the protein has either a positive charge (pH<pI) or negative charge (pH>pI) Runs requires higher voltages and longer periods of time, but gives r esolution up ±0.001 pH Dr Gihan Gawish 17

8. PREPARATION OF IEF GEL

9. A TYPICAL ISOELECTRIC FOCUSING GEL

10. Two Dimensional Electrophoresis (2-DE)

11. Three properties of proteins Size: molecular weight (utilized in 2-DE) Charge: pI (utilized in 2-DE) Hydrophobicity

12. What is 2-DE? Digest to peptide fragment MS analysis First dimension: denaturing isoelectric focusing separation according to the pI 2. Second dimension: SDS electrophoresis (SDS-PAGE) Separation according to the MW Interested spot

13. Only “Proteomics” is the large-scale screening of the proteins of a cell, organism or biological fluid, a process which requires stringently controlled steps of sample preparation, 2-D electrophoresis, image detection and analysis, spot identification, and database searches. The core technology of proteomics is 2-DE At present, there is no other technique that is capable of simultaneously resolving thousands of proteins in one separation procedure. Two dimensional electrophresis, 2-DE

14. Traditional IEF procedure: IEF in run in thin polyacrylamide gel rods in glass or plastic tubes. Gel rods containing: 1. urea, 2. detergent, 3. reductant, and 4. carrier ampholytes (form pH gradient). Problem: 1. tedious. 2. not reproducible. Evolution of 2-DE methodology In the past

15. Problems with traditional 1 st dimension IEF Works well for native protein, not good for denaturing proteins, because: Takes longer time to run. Techniques are cumbersome. (the soft, thin, long gel rods needs excellent experiment technique) Batch to batch variation of carrier ampholytes. Patterns are not reproducible enough. Lost of most basic proteins and some acidic protein. Evolution of 2-DE methodology OPERATOR DEPENDENT

16. Resolution for IEF: Immobilized pH gradients. Developed by Bjellqvist ( 1982, Biochem. Biophys Methods, vol 6, p317 ) PH gradient are prepared by co-polymerizing acrylamide monomers with acrylamide derivatives containing carboxylic and tertiary amino groups. The pH gradient is fixed, not affected by sample composition. Reproducible data are presented. Modified by Angelika Gorg by using thin film to support the thin polyacrylamide IEF gel, named Strips. ( 1988, Electrophoresis, vol 9, p 531 ) Evolution of 2-DE methodology

17. Run 2-DE, a quick overview

18. Run 2-DE step by step

19. Run 2-DE step by step

20. Total E. coli Proteins - 2-Dimensional Gel

21. 2-DE gel images of serum glycoprotein samples from the healthy and LC patients.  (A) Normal sample, (B) LC sample. The identified protein spots: (1) Anti TNF α antibody light chain (ATAL); (2) Chain L, structure of Fab D3h44 (D3h44); (3) Transthyretin (TTR); (4) AIM/CD69; (5) Alpha1-Antitrypsin (AAT); (6) Alpha2-HS-glycoprotein (AHSG) (7) Complement C3; (8) Zinc-alpha2-glycoprotein (ZAG); (9) Haptoglobin alpha2 chain (HpA2); (10) Ig heavy chain mu (BOT); (11) IGHM protein.