1. Lecture – FALL 2017
Protein Isolation and Quantification

3. How to isolate total protein
Lyse the cell
Solubilize the protein
For solubilize membrane protein, we have to use detergent in protein extraction buffer.
A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. 

4. The general detergent used in the protein extraction buffer
LYSIS BUFFER
Nonionic Detergents (milder)
Triton X100: Break lipid-lipid interaction and lipid-protein interaction
Anionic Detergent
SDS: Protein-Protein interaction
Sodium Deoxycholate: Protein-Protein interaction

5. Important Factors to be Considered When Choosing Lysis BufferpH
ionic strength
usage of detergent
preventative measure for proteolytic processes

6. Proteases inhibitor What are the proteases?
Upon lysis of the cell, proteases are released into the lysate
What are proteases?
Where are the proteases from when isolating the protein?
What are the proteases?
Enzymes that break peptide bonds between amino acids of proteins.
A protease (also called a peptidase or proteinase) is any enzyme that performs proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. Proteases have evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, fungi, bacteria, archaea and viruses.

7. Where are the proteases from when isolating the protein?
Animal cells: Lysosomes, contain a large variety of hydrolytic enzmyes that degrade proteins other substances.
Plant cells: Vacuole, many hydrolytic enzymes found in vacuole resemble those present in lysosomes of animal cells other organnells 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 the proteases.
The further optimize the results, we use the proteases inhibitors

9. Often used chemical protease inhibitors in protein isolation
EDTA: Chelating the Ca+2
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 specify.

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

11. UV absorption method
The amino acid tryptophan, tyrosine and phenlyalenine absorb light in the UV wavelength.
Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for protein containing Trp)

12. Disadvantages of UV absorption method
If some protein do not contain the amino acids, it will not absorb UV light.
Nucleic acid (DNA, RNA)contaminant will also absorb UV light.

13. Calorimetric 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
cheaper cuvette (cheap glass or plastic versus quartz quartz)
Not contamining absorbance from nucleic acid.

15. Colorimetric methods I: Bradford Method
A dye known as coomassie Brilliant Blue was developed for the textile industry. It was noticed to stain skin as well as the textile.
This dye (which normally absorbs at 465 nm) binds to the proteins and to absorb strongly at 595 nm)
The assay is sensitive but somewhat non-linear.

16. Lowery method A widely used method of measuring protein concentration.
A colorimetric assay.
Amount of the blue color proportional to amount of protein.
Absorbance read using nm light.
Lowery at all. 1951

17. Lowry Method Two reaction make the blue color develop:
Reactions of copper ion with the peptide bonds under alkaline conditions with the oxidation of aromatic protein residues.
Folin-ciocatleu reagent is used.
Concentration of the reduced Folin reagent is measured by absorbance at 750nm.

18. Making a strand curve with BSA (bovine serum albumin)
A graph that correlates Absorbance with protein concentration
Standard Curve generated by doing a Lowry Assay on protein solution of known concentration
Standard curve must be done each of the time unknowns are being tested

19. Standard Curve

20. Using Standard Curve

21. The SDS-PAGE

22. 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

23. SDS (Sodium dodecyl sulfate)

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

25. How about covalent link?

27. Heating your samples at 99C 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.

28. 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 protein by reducing them (adding hydrogen atoms).
Heating

30. Stacking Gel
To obtain optimum 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.

31. Gel Staining
Once protein fractionated by electrophoresis, to make them visible, staining with the 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.

32. Gel Drying Frame
It is important to remove all the air bubbles from between the two sheets of gel drying films.
Air bubbles may cause the gel crack during drying.
OCTAFRAME is a set of rigid plastic frames to support SDS-PAGE gels between two sheets of cellophane for drying on the bench. The drying is a few hours in gentle stream of air

33. Modern Technique: HPLC

34. Uses of HPLC
This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product stability and monitor degradation.
In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.

36. Picture of an HPLC column

37. WHAT AFFECTS SYSTEM Column Parameters Instrument Parameters
Column Material
Deactivation
Stationary Phase
Coating Material
Instrument Parameters
Temperature
Flow
Signal
Sample Sensitivity
Detector

39. Several column types (can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange

40. Normal phase
In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute.
For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample

41. Reverse phase
In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample.
Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water.

42. Size exclusion
In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinarily phase according to its molecular size.
Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules.

43. Ion exchange
In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase.
Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites.