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Electrophoresis Separation of the compounds (Protein) using electric…

- - - - A. Gradient gel: 4% to 15% using gradient mixer
        When to use:
        1- to resolve a broader range of protein sizes on one gel
        
        for sample is limited and cannot run multiple gels
        
        2- Gradients produce sharper bands
        
        leading edge encounter smaller pore sizes than lagging edge
        
        leading edge is passing through the gel at a slightly slower pace compared to the lagging edge resulting in a sharper band
        
        3- Can better separate similar-sized proteins - provides protein separation analysis with sharper, more discrete bands, and the longer the gel is run, the more distance (gap) between molecules traveling bands
      - B. Stacking gel & resolving gel
        Composition of gel layer
        
        variation affects formation of highly concentrated bands of sample in stacking gel and greater resolution of sample components in lower (resolving) gel
        
        acrylamide concentration determines the different pore sizes of the polyacrylamide gel
        
        protein trapped in between chloride +ve ion (front) & glycine -ve ion (back) forming a tight band
        
        Upper: Stacking gel
        
        to 'stack' all of the proteins into as narrow of a band as possible so that they all enter resolving gel at essentially same time
        
        lower acrylamide conc. (5%), bigger pore sizes
        
        HMW protein better resolve
        
        condition: -ve charged is disfavoured, low average electrophoretic mobility
        
        Lower: Resolving gel
        
        higher acrylamide conc. (10-15%), smaller pore sizes
        
        LMW protein better resolve
        
        condition: -ve charged is favoured, high average electrophoretic mobility
- - - - no dye front - electrophoresis was terminated too late, bands are crooked and the top of the resolving gel was uneven
        why: failure to use a properly overlay and on the right side, samples were overloaded
      - gel shows some field effects (curvature - lanes are not straight)- some overloading and some bands are crooked due to uneven resolving gel surface
      - smearing- an unevenly poured acrylamide mixture or due to gross overloading of protein
      - overloaded
      - tearing
      - frowning (run too hot)
      - bent bands
      - too low concentration
- - - - A. Contaminants removal procedure gambar
        
        Charged molecules (salt, buffer, nucleotides)- poor focussing, removal (dialysis, gel filtration)
        
        SDS (protein move to anode)- removal (acetone precipitation)
        
        DNA (gel pores-clogging, interfere with staining)- removal (DNAse/ sonication)
        
        particulate matter (gel pores-clogging)- removal (centrifugation)
      - B. Lysis Solution Composition for Proteins solubilization gambar
        
        Fresh & deionized urea: solubilize protein (8M-9M)
        
        Non-ionic/zwitter ionic (CHAPS,CHAPSO, NP-40) detergents- solubilize protein, maintain protein in solution during rehydration (20-100 mM)
        
        Reducing agent (DTT/DTE)- cleaves sulfide bonds (20-100 mM)
        
        Ampholytes (3-10)- maintain pH gradient (0.2-2%)
    - - Sensitivity (ng/spot) and advantages of protein staining
        
        Coomassie R-250 (50-100)- Simple,fast, consistent
        
        Colloidal Coomassie ( 5-10)- Simple, fast
        
        Silver stain (1-4)- Very sensitive, awkward
        
        Copper stain (5-15)- Reversible, 1 reagent negative stain
        
        Zinc stain (5-15)- Reversible, simple, fast high contrast neg. stain
        
        SYPRO ruby (1-10)- Very sensitive, fluorescent