Protein Isolation and Quantification

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Protein Isolation and Quantification
ABE Workshop 2007
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DNA
RNA
Protein
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How to isolate total protein

• Lyse the cell,
• Solubilize the proteins
• To Solubilize membrane protein, we have to use
  detergents in the protein extraction buffer

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The often used detergents in the protein
extraction buffer

• Nonionic detergents (milder)
• Triton X-100 break lipid-lipid interaction
  and
• lipid-protein interaction
• Anionic detergents (more denaturing)
• SDS protein-protein interaction
• Sodium Deoxycholate protein-protein
  interaction

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Proteases inhibitors

• Upon lysis of the cell, proteases are released
  into the lysate
• What are proteases?
• Where are the proteases from when isolating the
  protein?

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What are proteases?

• Protease (proteinases, peptidases or proteolytic
  enzymes) are enzymes that break peptide bonds
  between amino acids of proteins

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Where are the proteases from when isolating the
protein?

• Animal cells Lysosomes, contain a large variety
  of hydrolytic enzymes that degrade proteins and
  other substances
• Plant cells Vacuole, many hydrolytic enzymes
  found in vacuole resemble those present in
  Lysosomes of animal cells
• other organelles also have proteases

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How to prevent the proteins from degradation by
protease?

• the protein isolation is carried out at low
  temperature to minimize the activities of these
  proteases
• To further optimize the results, we use the
  proteases inhibitors

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Often used chemical protease inhibitors in
protein isolation

• EDTA (or EGTA) chelating the Ca2,
• PMSF a general serine protease inhibitor. It is
  the most common inhibitor used in protein
  purification. Soluble in isopropanol.
• The protease inhibitors cocktail a mixture of
  several protease inhibitors with broad
  specificity

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The protein quantification

• UV 280 absorption
• Colorimetric methods
• Biuret
• Lowry
• Bradford

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UV absorption method

• The amino acids tryptophan, tyrosine and
  phenylalanine absorb light in the UV wavelength
• Since the absorption is proportional to
  concentration, this is a useful way to
  quantitates protein concentration (for proteins
  containing Trp)

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Disadvantages of UV absorption method

• If some proteins do not contain these amino
  acids, it will not absorb UV light,
• Nucleic acids (DNA, RNA) contaminant will also
  absorb UV light,

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Colorimetric methods

• we can modify the protein sample with appropriate
  reagents so as to produce a color reaction and
  measure protein concentration using a
  spectrophotometer.

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Advantages of Colorimetric methods

• 1. Cheap cuvette! (cheap glass or plastic versus
  quartz quartz)
• 2. Not contaminating absorbance from nucleic
  acids!

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Colorimetric methods I Bradford Method

• A dye known as Coomassie Brilliant Blue was
  developed by the textile industry. It was
  noticed to stain skin as well as the textiles.
• This dye (which normally absorbs at 465nm) binds
  to proteins and to absorb strongly at 595nm.
• The assay is sensitive, but somewhat non-linear

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Lowry Method

• A widely-used method of measuring protein
  concentration
• A colorimetric assay
• Amount of blue color proportional to amount of
  protein
• Absorbance read using 500-750nm light
• Lowry et al, 1951

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Lowry Method

• Two reactions make the blue color develop
• Reaction 1
• Cu2 peptide bonds ? Cu1-peptide bond
  complex, produces purple-blue color
• Reaction 2
• Folin reagent Cu1-complex ? reduced Folin
  reagent, produces blue-green

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Making a standard curve with BSA (bovine serum
albumin)

• A graph that correlates Absorbance with protein
• concentration
• Standard Curve generated by doing a Lowry Assay
  
• on protein solutions of known concentration
• Standard Curve must be done each time unknowns
• are being tested

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The SDS-PAGE
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PAGE

• Gels are cast by polymerizing a solution of
  acrylamide monomers into polyacrylamide chains
• Gel pore size can be varied by adjusting the
  concentrations of polyacrylamide
• Smaller proteins migrate faster than larger
  proteins through the gel

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Native proteins
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SDS (sodium dodecyl sulfate) binds to and coat
the protein
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SDS
1. SDS disrupts some of the noncovalent
interactions that stabilize protein quaternary
and tertiary structures, facilitates
denaturation.  2. SDS also has a negative
electrical charge and binds to proteins in a
constant mass ratio of 1.4 1, so that the total
amount of detergent bound is directly
proportional to the molecular weight of the
protein.  3. The coating of negatively charged
SDS overwhelms the inherent charges of protein
molecules and gives them a uniform charge to mass
ratio.  4. This allows proteins to be separated
on the basis of their relative sizes,
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SDS

• all polypeptide chains are then forced into
  extended conformations
• SDS treatment eliminates the effect of
  differences in shape
• individual polypeptide chains migrate as a
  negatively charged SDS-protein complex through
  the porous polyacrylamide gel
• speed of migration is proportional to the size of
  the proteins
• smaller polypeptides running faster than larger
  polypeptides

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How about covalent link?
DTT/Me
SH
S-S
HS
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Noncovalent
covalent
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Heating the sample

• Heating your samples at 99ºC completed
  denaturation of the protein molecules, ensuring
  that they were in completely linear form. 
• This allowed SDS to bind all regions of each
  protein equally.

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Protein loading buffer

• Protein gel loading buffer contains Tris buffer
  to maintain constant pH
• glycerol to increase sample density,
• the strong ionic detergent SDS (sodium
  dodecylsulfate),
• ß-mercaptoethanol, a reducing agent. . 
  Beta-mercaptoethanol eliminates disulfide bonds
  in proteins by reducing them (adding hydrogen
  atoms).   
• Heating

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Running the gel
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Stacking gel

• To obtain optimal resolution of proteins, a
  stacking gel is poured over the top of the
  resolving gel.
• The stacking gel
• lower concentration of acrylamide (larger
  pore size),
• lower pH
• different ionic content
• This allows the proteins in a lane to be
  concentrated into a tight band before entering
  the running or resolving gel
• produces a gel with tighter or better separated
  protein bands

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Gel staining

• Once proteins have been fractionated by
  electrophoresis, to make them visible, staining
  with a material that will bind to proteins but
  not polyacrylamide. 
• the most common one staining with Coomassie
  Blue. 
• This is a dye that binds most proteins uniformly
  based on interactions with the carbon-nitrogen
  backbone. 
• The dye is dissolved in a solution that contains
  both methanol and acetic acid

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gel-drying framesfor drying of SDS-PAGE gels
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Gel drying

• SDS-PAGE gels between two moistened sheets of Gel
  Drying Film (from Promega) on the bench.
• Clamp the Gel Drying Frame
• Dry over night
• It is important to remove all the air bubbles
  from between the two sheets of gel drying films.
  Air bubbles may cause the gel to crack during
  drying

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References

• http//www.bio.davidson.edu/people/jowilliamson/Te
  chniques/Protocolweek5.html
• Lowry, O. H., Rosebrough, N. J., Farr, A. L., and
  Randall, R. J. (1951) J. Biol. Chem.193, 265275
• www.bio-itworld.com/ archive/091103/russell.html
  
• http//dwb.unl.edu/Teacher/NSF/C08/C08Links/pps99.
  cryst.bbk.ac.uk/projects/gmocz/gfp.htm

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Transfer

• In this procedure, a sandwich of gel and solid
  support membrane (Nitrocellulose or PVDF) is
  compressed in a cassette and immersed in buffer
  between two parallel electrodes.
• A current is passed at right angles to the gel,
  which causes the separated proteins to
  electrophorese out of the gel and onto the solid
  support membrane

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Transfer the protein from the gel to the membrane

• Transfer of the proteins fractionated by SDS-PAGE
  to a solid support membrane (Western blotting)
  can be accomplished by electroblotting