1. Protein Isolation and Quantification
ABE Workshop 2007
Protein Isolation and Quantification

2. DNA
RNA
Protein

3. 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

4. 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

5. 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?

6. What are proteases?
Protease: (proteinases, peptidases or proteolytic enzymes) are enzymes that break peptide bonds between amino acids of proteins

7. 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

8. 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

9. 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

10. The protein quantification
UV 280 absorption :
Colorimetric methods:
Biuret
Lowry
Bradford

11. 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)

12. 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,

13. 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.

14. Advantages of Colorimetric methods
1. Cheap cuvette! (cheap glass or plastic versus quartz quartz)
2. Not contaminating absorbance from nucleic acids!

15. 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

16. 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 nm light
Lowry et al, 1951

17. 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

18. 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

21. The SDS-PAGE

23. 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

24. Native proteins

25. SDS (sodium dodecyl sulfate) binds to and coat the protein

26. 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,
SDS

27. 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

28. How about covalent link?
DTT/Me SH
S-S HS

29. Noncovalent
covalent

30. 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.

31. 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),
β-mercaptoethanol, a reducing agent. .  Beta-mercaptoethanol eliminates disulfide bonds in proteins by reducing them (adding hydrogen atoms).   
Heating

33. Running the gel

34. 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

35. 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

36. gel-drying frames for drying of SDS-PAGE gels

37. 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

38. References
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.193, 265–275
archive/091103/russell.html

39. 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

40. 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