We've seen a Cell's Activity requires a protein, so how do we study what they can do?
Protein Biochemistry - Some Techniques & Methodolgies for Isolating Proteins
Some Online links to protocols, Methods and Procedures:              Methodologies  &  Extraction  &  Purification  &  Protein-Chemistry-Journals                                 Cell Biology Protocols and a CMB Lab Manual                                         some-not-so-precise protocols
Isolation & Purification of a "PROTEIN"...
is based upon the chemical & physical properties size,   shape,   charge,   solubility of the protein one wants to isolate
review pages ecb/6e: Panel 4-3,4,5, & 6   pg170-174

Protein Methodolgy in the Science of Biological Chemistry...               Know the Basics,  master the Method, be Persistent...   and  repeat, repeat, repeat.
	Protein Isolation Techniques
	1st:     Selection of appropriate specimen for protein isolation... next:   Fractionation & isolation methodologies for 'soluble proteins'...            by using cell & tissue homogenization to break apart cells - methodology*                                                Grind & Find          i.e., mortar/pestle, homogenizers, sonicators to break open cells.
homogenize (break apart) cells in...              mortar&pestle,   tissue grinders*, homogenizers,             FastPrepHomogenizer,   cell disruptors,             blendors (Waring*), and barocyclers ...                                    or  in beaters or sonicators* in an osmotically,               buffered medium with an enzyme's substrate              &/or in an isotonic media on ice*          How to choose a cell disruption method animation

      results...   ruptured cells produce a  liquified cellular homogenate

 
FRACTIONATION of the HOMEGENATE...
                  
1. CENTRIFUGATION - applies centrifugal force to separate homogenate into its components
        producing a pellet (solid part) & supernatant (liquid portion) :  pellet/supernatant*
                     - 1924    T.Svedberg (1926 Nobel Prize) invents Analytical Ultracentrifuge 
                          - 1938    G. Beherens isolates nuclei by centrifugation
                          - 1946    George Palade, Hogeboom & Schneider perfect differential centrifugation in sucrose  
                          - 1955    C.de Duve isolates lysosomes (1st organelle not defined by microscopy)
    2 major types:   differential centrifugation   vs.    rate-zonal        (panel 4-3*)
               - speeds from 100 x g to 1,000,000 x g       clinical centrifuges - Sorvall Lynx 6000 fuge
                                                                                    ultracentrifuge - pic*   &    rotor failure*
   examples of separation protocols:
   - via  differential centrifugation   ecb panel 4.3*
                                repeated centrifugations at increasingly higher speeds
                                separates organelles by their SIZE and DENSITY
                                                              1000g  =  whole cells & nuclei,       20,000g  =  mitochondria/chloroplasts,  
                                     80,000g  =  vesicles                         100,000g  =  ribosome, Viri, etc...
 
   - via  rate zonal - separation by shape/conformation/mass over a shallow gradient
                                         =  gradient preparation*   &  velocity sedimentation*
   - or  by buoyant density    =  equilibirum sedimentation - buoyant density gradient centrifugation*
     
                 comparison of     =  shallow [a]   or   steep gradient [b]*
                     >    summary: cell fractionation & centrifugation animation*^view@home      
 

   
 PROTEIN SEPARATIONS...  proteins can separate based upon their physical properties-
                                                   size,   charge,   affinity for ligands,   shape,   etc…

Column Chromatography...             principles*            sample columns       done in cylindrical glass column, on permeable support media or matrix,       which retards flow of selected molecules, while others pass through.

        Kinds of glass column chromatography:   
   
                 ion exchange chromatography… charged ligands
                          separates molecules with differential charges -     ecb4.4c*
                          column matrix retards passing proteins of opposite charge  
                                            DEAE cellulose   [diethylaminoethyl cellulose]  (+)
                                            CM-cellulose       [carboxymethyl cellulose]       (-)
   
                 size exclusion chromatography...    [gel filtration chromatography]                 
                          which separates on the basis of molecular size - ecb4.4c*
                          inert "sized" media (beads) retards smaller size proteins...  

                 affinity chromatography
                          based on biological activity, an inert media polymer with a ligand
                          (antibody, enzyme substrate) binds a specific protein  - ecb4.4c*   

                  Review of Types:   ecb/6e  - Panel 4.4 page 172*^review  &    MCB-7e^review                                                                                                              

 
  Separation of Proteins by electric charge   =    Isolation and Visualization...  
  proteins migrate in an electrical field at rates that depend upon their net charge, size, and shape

  ELECTROPHORESIS... separation* of proteins in electrical field & on a support media...
     
       PAGE*: in a media as porous gel (polyacrylamide/starch), which separates protein molecules
                       on the basis of size, conformation, and net charge   gels stained*
    
          Isoelectric focusing* (IF): proteins are separated in a gel of a continuous pH gradient... 
                              proteins move to point in gel equal to its pI [isoelectric point], i.e., no net charge
    
          SDS-PAGE* (SDS): Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis...
                              proteins treated with ionic detergent separate according to their size
                              SDS binds to protein @ 1 SDS/2 aa's thus proportional to a protein's MW

       
          2D-electrophoresis: is a combination of IF & SDS together (mcb3.36a*)    
                                                see a protein fingerprint... PEPTIDE MAP*       2D-PAGE of Human Peptides   

How does one Identify a protein’s presence in samples   &   its quantify its amount...        How do we know that we actually have protein in our 100,000 x G supernatant???
Identification - is often done by  Spectrophotometry         spectrophotometers measure intensity of light beams before & after        light passes through a liquid solvent with sample dissolved in it,  (in a cuvette-pic)...        compares the two light intensities over a range of wavelengths.           figure*                        Percent transmittance...                             ratio of intensity of light passing through the sample                to the intensity of light shining on sample multiplied by 100%.        Absorbance...                 is the log of the transmittance                a plot of absorbance vs. wavelegth - gives an absorption spectra*                   sample instruments...    Spectronic 20   &   Beckman DU640 UV-Vis

 

             

 

 

   
 SPECTROPHOTOMETRIC  METHODS  of  QUANTIFYING  PROTEINS

   UV absorbance at 280 nm measures aromatic aa's presence - (figure*) 
        
   Colorimetry reactions - colored dye do bind to amino acids including...
          Ninhydrin reaction - rx's w amines = blue color  (10^-12 M) (Ninhydrin test CSI*-reference)
          Biuret* test = measures mg protein quantities…   based on Copper ion
                                binds stiochiometrically = violet color  how to prep standard curve
          Bradford test = measures ug protein amounts  [range 0 µg/mL to 2000 µg/mL]
                                based on dye Coomassie blue - binds to peptide   Bio-Rad* Bradford test

   
    Quantification of amounts of protein present...
               quantification is based on Beer-Lambert Law which results in a...         
               a linear relationship between... light Absorbance vs. Concentration  (figure)
                                           Protein Standard Curve  (figure*)
               top 3 most cited papers in literature are on methods of protein quantification.              
 

  

       CORRELATION  of  PROTEIN CONCENTRATION  with  ENZYME ACTIVITY
                                      relating protein amounts to enzyme activity
 
         1  (international)  UNIT of  ENZYME ACTIVITY…
                    that amount of protein which converts 1 micromole of substrate
                    to product per min at 25⁰C at optimal pH
                                 Hexokinase - 1 unit (I.U.) will phosphorylate 1.0 µmole 
                                   of hexose sugars per minute at pH 7.0 at 25°C   
  

         1  UNIT of  SPECIFIC ACTIVITY… 
                    the number micromoles [IU] converted per min  per mg protein
                                   i.e., Units (as above) of enzyme activity per mg protein
                                          10 umol/min with 2.5 mg protein = 4 IU/mg 
     

         1  UNIT of  MOLECULAR ACTIVITY…  
                                 number of units [IU]  of enzyme activity per µmole of enzyme

 

An Example of a Protein Purification Sequence...

Purification Table for a "NEW" Enzyme [Horse Radish Peroxidase] substrate-red + H2O2  <-->  substrate-ox + 2H2O
STEPS	Fraction Volume (ml)	Total Protein (mg)	Enzyme Activity (units*)	Specific Activity (units/mg protein)
1.  Homogenate	1,400	10,000*	100,000*	10
2. 10,000 x g       supernatant	280	3,000	96,000	32
3. Ion-exchange       chromatography	90	400	80,000	200
4. Gel filtration       chromatography	80	100	60,000	600
5. Affinity       chromatography	6	3	45,000	15,000**
*    1 unit of activity = 1.0 micromoles H202 --> H20 & H+ per min        **  1,500 fold purification increase of peroxidase over homogenate
	End	material	exam #3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SKIP the MATERIAL BELOW

 

 Table Common Isotopes and  Nuclides,

SKIP material below:

      high pressure liquid chromatography  (Varian HPLC)
               HPLC has been used to detect vitamin levels in blood serum,
               detect drugs in urine, and separation of complex biologicals.
   
               a sample is vaporized and  a liquid presures solvent is passed to a HPLC column,
               through a HPLC column of stationary absorbent material* under high pressure;
               The sample moves through a column, which separates mixture
               into compounds according to their affinity for the stationary phase
               and retention time held to HPLC column.
             sample HPLC chromatogram*    &     HPLC applications open applications link

    Skip the pages BELOW... you are not responsible for it.

2 useful procedures of Proteomics commonly used to help determine peptide structures:
  
  1.  Mass Spectrometry: (mcb3.40*)  detects exact MASS of small peptides [MW]
            - a purified protein is treated with trypsin to produce peptides
                          [trypsin cleaves polypeptides on COOH side of LYS & ARG residues]
            - peptides are dried onto metal plate, blasted with laser, vaporizing them as peptide ions*,
            - peptide ions flow through electric field & the time it takes to pass a detector
                     is a function of their charge & mass  which can be related to genomes.  (ecb fig 4.11)
             - individual cell mass spectroscopy* via nanotechnology
 

   2.  X-Ray Crystallography: (mcb3.42*) determines 3-D shape of molecules mathematically.
            - 1st crystallize a purified protein (a large, highly ordered, conformational array)
            - atoms within crystal scatter a beam of X-rays  forming a diffraction pattern on film
            - up to 25,000 spots; computer program interprets patterns atom structure. (ecb fig 4.12)
  
    

 

CHROMATOGRAPHY...       (Color Writing)          - J.Chromatographic Science                separation of molecules based on differences in their structure &/or             physical properties when interacting with a stationary support media.

PARTITION chromatography.... developed by R.L.M. Synge                       small MW molecules are partitioned between phases of                        2 different solvents (water/alcohol) on a support media            PAPER chromatography... uses cellulose as support media [chlorophylls*] THIN LAYER chromatography... media is silica gel on glass plates/columns                                                                        chlorophylls on thin layer*                      DNA Electrophoresis  can also separate polynucleotides by their charge (pic) [video]     Fluoroescamine dye = pg quantities of protein in sample solutions...  (10-12 M)                                          SDS-PAGE animation*view@home