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Chapter 10: Analysis of proteins

Chapter 10: Analysis of proteins. Purification schemes: 1. soluble recombinant proteins 2. insoluble recombinant proteins that are produced as inclusion bodies in the cytoplasm of host cells. 3. soluble proteins present in the natural host cells

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Chapter 10: Analysis of proteins

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  1. Chapter 10: Analysis of proteins

  2. Purification schemes: 1. soluble recombinant proteins 2. insoluble recombinant proteins that are produced as inclusion bodies in the cytoplasm of host cells. 3. soluble proteins present in the natural host cells 4. membrane-associated and poorly soluble proteins (non-recombinant).

  3. Protein concentration determination methods: 1. A280 (UV lamp) if A is > 2, dilute the sample. 2. Bradford method (Bio-rad) Stain protein with Commassie blue solutions, and read A595.

  4. Quantitative amino acid analysis A good way to check purity and confirmation of the protein sequence. Protein sample is hydrolyzed by 6M HCl, then Beckman 6300 system for ninhydrin detection. Frequently amino acid residues in a protein: Gly, Ala, Ser, and Leu. Rare amino acid residues in a protein: Trp, Met, His, Cys, and Arg. Calculate ratio of each aa (Ratio1) Ser, and Thr (loss rate is high in the method) and rare aa (His, and Met) are taken out from the total amounts, re-calculate the ratio (Ratio 2).

  5. Safety elctrophoresis operation: To start: Power turn to zero Turn on power Hook gel apparatus (use one hand with one outlet at a time, the other hand is free. ) Set to fixed current, but sometimes fixed voltages, and run . To stop: Power turn to zero Power off Unhook gel apparatus (use one hand with one outlet at a time, the other hand is free.) DO NOT unhook gel apparatus first, before turn the power off. Otherwise, electrical charges stays in power even the power is off and will cause shock.

  6. Polyacrylamide gel elctrophoresis (PAGE) Laemmli method: two buffer systems, discontinuous Denature PAGE: SDS-PAGE Sample proteins are solubilized by boiling in the presence of SDS. 2-Me and DTT are added during solubilization to reduce disulfide bonds. SDS (detergent with polar head and non-polar tail) for coat protein with negative charge. Protein is denatured. - Negative charge density is same with different proteins. - Negative charges are proportional to the MW of the proteins.

  7. Denature PAGE: SDS-PAGE 1. Tris –glycine buffer for separation of MW > 14 kd. (because SDS move to the 14 kd and below area) 2. Nonurea peptide separations with Tris buffer. Increased buffer concentrations for separations to 5 kd. 3. Tris – Tricine buffer. for separation of MW of 10-15 kd

  8. Non- Denature PAGE No boiling, SDS, 2-ME or DTT, others are all same. (Some proteins will move to anode and into buffer, the pH may need to be changed.)

  9. 2-D gel electrophoresis 1-D: isoelectric focusing use Bio-Rad Tube Cell 175, or tube cell adaptors with mini-protean III. Prepare Gel with ampholytes, will generate pH gradients when electrophoresed. Proteins will stay at pH position equivalent to its PI. commercial strips are available. NEPHGE (non-equilibrium pH gradient electrophoresis) Modifications can resolve very basic or acid proteins. 2-D: SDS-PAFE

  10. Staining proteins in gel 1. conmmassie blue staining non-specific binding to proteins, detection limit: 0.3 to 1 μg/ protein band. 2. silver staining (S-H group, and C=O group) some proteins cannot be stained. detection limit: 2 to 5 ng/ protein band nonammoniacal silver staining detect proteins that cannot proteins by 2. 3. Fluorescent staining SYPRO red, ruby or orange See under 300nm UV illuminator, or a laser scanner. Staining procedures for IEF, native gel and SDS-PAGE might be different.

  11. Immunoblotting Tank transfer Semi-dry (a protocol for blotting of a CB-stained gel is provided) Ponceau S can reversible stain the membrane. CB stains permanently, cannot do further hybridization.

  12. Immunodetection 1. directly conjugated secondary antibody (eg. AP or HRP-conjugated) 2. Avidin-biotin coupling to secondary antibody Visualization with chromogenic substrates HRP- or Ap-based. with luminescent substrates HRP- or AP based.

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