1. Why Do We Analyze Proteins?
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.

2. Main Steps in Protein Analysis
Extraction of proteins.
Purification of the protein.
Structural Characterization of the protein.

3. Protein Purification Protein Property Technique Solubility
Q Proteins can be purified according to certain properties they possess. These properties allow us to employ different techniques in purifying proteins.
Protein Property
Technique
Solubility
Differential Precipitation
Molecular Size
Gel Filtration Chromatography
Molecular Charge
Ion Exchange Chromatography
Hydrophobicity
Reversed Phase HPLC
Biological Activity
Affinity Chromatography

4. Chromatography
Chromatography basically involves the separation of mixtures due to differences in the distribution coefficient of sample components between 2 different phases. One of these phases is a mobile phase and the other is a stationary phase.

5. Gel Filtration Gel permeation chromatography
Size exclusion chromatography
Separation of molecules on the basis of size (and shape)

6. Gel-Permeation Chromatography
Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.

7. How it works: A column is packed with a semi porous material
glass beads
glass wool
something that doesn’t dissolve in the solvent carrying the polymer chain
In some cases simple soap detergents like Tide are used

8. How does the column work?
Hydrodynamic radius (Stoke’s radius)
Hydration
Shape
Column resin – polymer beads. Porous beads. Based on size. When we talk about size of a molecule tumbling in aqueous solvent, there are two things to consider: its hydration and shape. Hydrodynamic radius or the Stroke’s radius takes both into account.

9. Gel Permeation Chromatography
Gel permeation chromatography (GPC) is used to make reasonable estimates of the molecular weight distribution of polymers
For GPC work to be reproducible you must have control over:
flow rate
temperature
solvent composition
tunnel size of the stationary particles (it must not change)
other reasons may be added later...

10. Partial Structure of Sephadex

12. Matrix Types Material Sephacryl Protein (kD) Dextrans (kD) S-100 1-100
5-250
1-80
S-300
2-400
S-400
S-500
40-20,000
Material
Sephacryl
dextran
Sephadex
Sepherose
agarose
Superdex
mixture

13. Solvents Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane

14. Detector Ultraviolet Detector 200-400nm 254 nm
Reflective Index Detector Universal Detector

15. Column Parameters Vo = void volume Vt = total volume
Vs= volume of solvent held in the pores. This is normally approximated to
Vt-Vo = volume of beads
Vo = Elution volume of a large “totally excluded” molecule such as blue dextran
Vt = Physical volume of column

16. Resolution
Resolution proportional to square root of column length. Also affected by rate at which column is run

17. Resolution

18. Factors Increasing Resolution
Increase column length
Decrease column diameter
Decrease flow-rate
Pack column uniformly
Use uniform stationary phase (packing material)
Decrease sample size
Select proper stationary phase
Select proper mobile phase
Use proper pressure
Use gradient elution

19. Practical Stuff Column packing
Sephadex G50
fractionation range of 2 to 30 kDa
Column buffer: Phosphate buffer at pH 7.0
What we will be loading into the column
Blue dextran
Cytochrome C
Potassium ferricyanide

20. Elution Profile Ve
Ve = Elution volume (volume of solvent between injection and elution). Dictated by proportion of porous matrix available to molecules (Kd).

21. Design of Column Column size Solvent Matrix Material Pore size
Analytical or preparative
Solvent
Inert matrix most solvents OK
Matrix
Most important consideration
Many different types
Material
Pore size

22. DO NOT RUN DRY!! Setting up the column
Add 10 ml column buffer to column to check tubing and leakage
=> what happens when liquid is inside an open-ended column?
DO NOT RUN DRY!!

23. Matrix Types Material Sephacryl Protein (kD) Dextrans (kD) S-100 1-100
5-250
1-80
S-300
2-400
S-400
S-500
40-20,000
Material
Sephacryl
dextran
Sephadex
Sepherose
agarose
Superdex
mixture

24. Pouring the Column

25. Packing the Column
Let resin form 1-2 cm bed and then start buffer flow. Adjust to ml/min (no faster!).
Collect buffer in a clean beaker to be reused later.

26. Packing the Column
The column height should be at least 25 cm at this point.
Watch for any bubbles in resin before it settles all the way

27. Adding Additional Resin if Needed
Make sure it’s more than 25 cm. it will settle down.
- Gently make a vortex in buffer above resin to create a slurry
Add extra resin down glass rod and let it compact
Don’t let the column run dry. EVER

28. Fraction Collector Calibration
Measure volume of 150 drops to determine number of drops that will give 1.5 ml fractions

29. Running the column Sample size / Fraction size Running time
0.5 – 5% of total bed volume (Vt).
Concentration limited by viscosity
Running time
Determined by “trial and error”
Slow rates allow efficient partitioning into pores and thus increase resolution
Slow rates increase diffusion of sample on column thus increasing peak width and reducing resolution.
Protein about 5 mL cm-2. h-1

30. Loading Sample
Make sure the buffer goes down all the way into the column but do not dry it!!! How can you do that?
Ooze sample on gently with pipet to avoid disturbing the resin
Start collecting buffer in an empty beaker immediately

31. Adding Buffer Head and Reservoir Flask
What is reservoir flask doing?
Why do you need buffer head?
Add 5 cm buffer head gently, and then connect Mariotte flask to maintain buffer head
Keep collecting buffer eluate in the beaker

32. Determining the Void Volume with Blue Dextran
Band will broaden as it filters down column
Keep collecting buffer eluate in beaker until band nears the bottom

33. Collecting Blue Dextran Fractions
Start collecting fractions as Blue Dextran nears bottom of column
Save eluant collected before first fraction, and measure volume in graduated cylinder.

34. Absorbance Measurement
Save all column fractions.
Dilute each tube with 2 mL of phosphate buffer.
Add 4 mL of phosphate to cuvette to calibrate the spec 20, then measure all the absorbance in order.
Repeat the measurement for the overlapping tubes but change wavelength

35. Determination of Molecular Weight
Calibrate column with known standards
Plot Kav vs log MW

36. Summary
Each group will receive 100 mL of blue dextran, 150 mL of cytochrome C and 70 mL of potassium ferricyanide.
Turn on the switch to let the buffer drip until the top buffer is almost gone, then pipet the mixture of sample to the top of column evenly.
use a beaker to collect all the buffer from the bottom of the column, when the sample layer is empty, add 1 mL of buffer to the top, repeat the process 3 to 5 times until the top layer is colorless, then add at least 3 cm length of buffer on top of the column and connect the column to a buffer reservoir.
Take a test tube and mark a 1 mL line by transfering 1 mL of solution to it. Then count the number of drops of the buffer to fit exactly 1 mL volume, then pour the buffer back to the beak.
When blue dextran is approaching the bottom of column, switch beaker to test tube and collect 2 mL of buffer to each test tube until all the color bands drain out of the column. Meanwhile, measure the volume of buffer collected in the beaker by a graduate cylinder.
add 2 mL of buffer to each test tube, and measure the absorbance of each fraction at relevant wavelength. Be sure to repeat the overlapping tubes.

37. Requirements for Lab Report
Lab report should have a table to show all the absorbance of each tube, as well as the overlapping absorbance
Should include a figure to correspond the absorbance to tube number (or elution volume), whichever is good for you.
should have a figure to show the relationship between the elution volume and log MW.