Protein Chemistry - Techniques & Procedures for Isolating Proteins

Methodologies,  Techniques,  &  Protein Isolation Procedures
Protein Chemistry Journals   &  books

 
Isolation & Purification of a "PROTEIN"...

3' --> 5' exonuclease of DNA Polymerase I

is based upon its chemical & physical properties
size, shape, charge, solubility 

Cell Biology Protocols & Methods
LAB MANUALS

                                     reading pages 92-108; 391-394

 
 
  
     

 

 

 

 

Protein  Isolation  Techniques                 

Fractionation & Isolation Methodologies for Cellular Molecules...  
  

    uses mortar/pestle, homogenizers, sonicators to break open cells
n    
 
  cell & tissue homogenization
   -  methodologies*
    grind cells in... 
         mortar & pestle,   tissue grinders,
         homogenizers,   cell disruptors,  
blendors (Waring)
         and barocyclers ...                                            --->

         or in beaters or sonicators in an osmotically, 
         buffered medium w enzyme's substrate
         &/or in an isotonic media 
on ice*

      results...   ruptured cells producing a liquified  cellular homogenate

 

 

 

 

 
 
centrifugation - applies centrifugation force to separate
                
     produces a pellet (solid part) & supernatant (liquid portion) :  pellet/supernatant*
                                

       
- 1924    T. Svedberg invents analytical ultracentrifuge
       
- 1938  
 G. Beherens isolates nuclei by centrifugation 
       
- 1954    C. d
eDuve isolates lysosomes  (1st organelle not defined by microscopy) 

  

   
2 major types:   differential centrifugation (mcb3.34a
*)  &  Rate-Zonal (mcb3.34b*)                                          speeds from 100 x g   to    600,000 x g
                
                             clinical centrifuges     -    fixed angle vs. swinging bucket
                     
                        ultracentrifugepic        failed rotors
     

              -results:  by   differential centrifugation  
mcb fig9.25*
                                        repeated centrifugations at increasingly higher speeds
                                        separates
organelles by their mass and density
                                          
                300g   =   whole cells & nuclei,
                                             
              12,000g   =   mitochondria/chloroplasts
                                            
             
100,000g   =   microsomes, ribosome, etc...

                         & 
  by size  velocity sedimentation* in a shallow sucrose gradient [a]
 
                 &   by density... equilibirum* sedimentation in a steep CsCl gradient [b] 
                                                                  
       Sumanas, Inc. cell fractionation animation*       
 

 

 

 

 
  CHROMATOGRAPHY...       (Color Writing)          journal - JCS   
                                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 [Analtech & Varian]
                                                                                                                
(pics)

                 

 

 

 

 

 

 
 
PROTEIN SEPARATIONS...  Proteins are separated 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 column chromatography:   -    3 types  (pg 97)
                 ion exchange chromatography charged ligands
                                  separates molecules with different net charges - mcb3.37b*
            
                      column matrix retards passing proteins of opposite charge  
                    
                            DEAE cellulose   [dimethylaminoethyl cellulose]    (+)
                                 
                CM-cellulose       [carboxymethyl cellulose]         
(-)  [figure]
   
                 size exclusion chromatography...    gel filtration                                     [figure]
                                  which separates on the basis of molecule size - mcb3.37a*
 
                                inert "sized"
media (beads) retards smaller size proteins...         [figure]
                                                                                                                                            ecb panel 4.4

 

 

 

 
   
 

  

      affinity chromatography
                based on biological activity, an inert
media polymer with a ligand
               
(antibody, enzyme substrate) binds a specific protein    - mcb3.37c*

    molecular imprint polymer chromatography
               manuafcture of specific shaped and contoured chromatography media
               for high yield isolation of solutes                                     
review of types

     
high pressure liquid chromatography  (Varian HPLC)
               a sample is vaporized and injected in to a HPLC column,
              
containing stationary liquid phase under high pressure;
               sample moves through a column and separates mixture
               into compounds according to their
affinity for the stationary phase
               and
retention time held to HPLC column.
                            HPLC chromatogram
*    &     HPLC applications open applciations link

 

 


 

 


 Visualization  &/or  Isolation  of a protein...  
    
proteins migrate in an electrical field at rates that depend upon their net charge, size, and shape

 
Gel Electrophoresis...   separation by charge (separation of* in a media as  gels* )   (pg 94)
                                   
PAGE*
: in a media as porous gel (starch/polyacrylamide) gels & staining
    

          
Isoelectric Focusing*: proteins are separated in a gel of a continuous pH gradient
                                          
proteins move to point in gel equal to its pI, i.e., no charge
    

          
SDS-PAGE*: Sodium Dodecyl Sulfate - polyacrylamide gel electrophoresis...
                              proteins treated with
ionic detergent* that separates according to size
                             
             SDS binds to protein @ 1 SDS/2 aa's thus proportional to a protein's MW
          
2D-electrophoresis: a combination of IF & SDS together   (mcb3.36a* & result* )   
                                
   see
PEPTIDE MAP [a protein fingerprint]...  2D-PAGE Human Peptide Maps
 
    
 DNA Electrophoresis*: separates polynucleotide strands by charge (pic)      ecb panel 4.5


 

 

 

 

 

 

PROTEOMICS -
           is the science of protein expression of all the proteins made by a cell.

Proteome - all proteins being made according to the transcriptome (RNA profile).
                
- often visualized by a system interaction map as seen in the proteogram*
                    of some 3,500 proteins & their interactions in a Drosophila cell

      Human Proteomics Initiative      HPI 
                   
a highly curated database of human protein sequences:

                          Commercial Proteomics Equipment & Supplies & other techniques                                             Proteomics & Drug Therapies  &   Proteomics Symposia
 
    

 

 

 

 

3 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)
 
   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; a computer program interprets the patterns atom structure.
(ecb fig 4.12)
  
   3. 
NMR Spectroscopy : magnetic signal indicate distances between atoms.
            - nuclei of atoms are "magnetic"... and magnetism is influenced by adjacent atoms
            - solution of protein is placed in strong magnetic field; then bombarded with radio waves
            - hydrogen nuclei generate NMR signals (NMR spectrum
*) indicating distances between atoms
      - allows computation of 3D structure of molecule.

 

 

 

 

 

 

 
Identification of protein’s presence & &  its quantification...

  Identification - is often done by  spectrophotometry    
  
   
spectrophotometers measures intensity of light beam before & after
       light passes through a liquid solvent with sample dissolved in it, 
       (in a cuvette)... 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                         absorption spectra
*
  

    instruments... Spectronic 20   
a neat experiment by Gary Betrand - U.Missouri Rolla

 

 

             

 

 

   
 
SPECTROPHOTOMETRIC  METHODS  of  DETECTING  PROTEINS

   UV absorbance at 280 nm. (measures aromatic aa's -  figure*)    
  
Colorimetry reactions - colored dye binds to amino acids
      
  
Ninhydrin reaction - rx's w amino = blue color
(10-9M)        (CSI
+ fingerprint)
         
Biuret* test = mg quantities…   based on Copper ion
  
                            
binds stiochiometrically = violet color
  how to prep standard curve
          Bradford test = ug amounts
  
                            
based on dye Coomassie blue - binds to peptide   
Biorad Bradford test*
          
Fluoroescamine dye = pg quantities..
(10-12 M)
   
    Quantification of amounts of protein present
               Quantification is based on BEER-LAMBERT Law        
                linear relationship between... light Absorbance vs.
Concentration
  (figure)
                                       
  Protein Standard Curve 
(figure*) 

  

 

 

 

  
 
Quantification  of Protein Concentrations with ENZYME ACTIVITY
                                                   relating protein amounts & enzyme activity
 
 
        1  
(international)  UNIT of  ENZYME ACTIVITY…
                    that
amount of protein which converts 1 micromole of substrate
                    to product per min at 25
0C at optimal pH
                       
          UREASE - 1 unit (IU) will liberate 1.0 µmole of ammonia from
                                   urea per minute at pH 7.0 at 25°C
   [equivalent  to 1.0  I.U.]  
  
 
        1  UNIT of  SPECIFIC ACTIVITY… 
                    the number micromoles converted per min  per mg protein
                                   i.e.,
Units (as above) of enzyme activity per mg protein  
     

         1  UNIT of  MOLECULAR ACTIVITY…
                           
     number of units of enzyme activity per µmole of enzyme
 
                                                                                                 
 .              .


 

 

 

 

    An Example of a Protein Purification Sequence...

Purification Table for a "NEW" Enzyme
[Horse Radish Peroxidase]
not previously isolated nor purified. 

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.  Precipitate 280 3,000 96,000 32
3.  Ion-exchange
     chromatography
90 400 80,000 200
4. Gel filtration 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 of peroxidase                                                     

 

 

 

 

 

 

 

      The Material for this section ends with the table above
     do not use the material below.

 Radioisotopes      
 Table Common Isotopes*
 Nuclides, Nuclear Medicine & Los Alamos

Isolation Protocols,  

blackwell

ecb panel 4.3