1. Protein Seperation Methods

2. Protein techniques Protein Identification Protein Expression
Protein Purification
Protein-Protein interactions
Application in literature

3. Protein Identification
Sequencing (Edman degradation)
Determine approx the first 20 AA
Centrifugation (cellular location)
1D/2D Gel Electrophoresis
Mass spectrometry
Break sample into peptides
Molecular mass is determined using mass-to-charge ratios of ions
AA sequence can be determined

4. Centrifugal separation
Differential centrifugation
Sediment coefficient (mass, density and shape)
Crude separation of cell fractions
Rate Zonal (mass)
Density of particles > density of solution
Separation based on rate of sedimentation
Time sensitive
Isopycnic (density)
Density of particles < highest density of solution
Separation based on reaching equilibrium position in density gradient
Time in-sensitive

5. Differential centrifugation

6. Gradient centrifugation

7. Gel electrophoresis Denaturing – SDS-PAGE Non-denaturing
SDS gives uniform neg. charge
Separates proteins by size/mass
Non-denaturing
Separates based on charge and size/conformation
Often combined with Western blotting (using antibodies specific for proteins of interest)

8. SDS-PAGE

9. 2D gel electrophoresis 1st dimension 2nd dimension
Separation based on pI
isoelectric focusing of zwitterions
2nd dimension
Normal SDS-PAGE

10. 2D-GE

11. Difference Gel Electrophoresis (DiGE)- quantitative comparisons

12. Mass spectrometry IDs based on mass-to-charge ratio
Samples are broken down and analyzed
Proteins -> peptides
Able to determine seq of peptides
Database search to ID protein

13. Mass spectrometry

15. Mass spec combos LC/MS MALDI-TOF MS Tandem MS
Liquid chromotography to separate peptides
MALDI-TOF MS
Matrix-assisted laser desorption-time of flight
Samples are ionized and “flight time” through an electrified tube is measured
Tandem MS
Multiple MS measurements on a single sample
Identifies peptide sequence

16. Protein Expression and Purification
Why?
Obtain pure (clean) protein
"Don't waste clean thinking on dirty enzymes“
- Arthur Kornberg
Powerful experimental tool
Simplifies the system in which you are asking a question
Confirmation of a hypothesis that is developed in a more complex system
Arthur Kornberg "Don't waste clean thinking on dirty enzymes"

17. Protein Expression Why over-express the protein?
Make large quantities to facilitate purification/study
Analyze biochemical properties
Perform structural analyses
Crystallization
NMR
Identify protein interactions
Make Antibodies

18. Expression systems E. coli Yeast Insect cells In-vitro systems
Prokaryotic expression workhorse
Yeast
For bacterial or eukaryotic proteins
Large amounts of protein
Insect cells
Post-translational modifications
In-vitro systems
wheat germ, rabbit reticulocyte

19. Purification strategies
Exploiting protein chemistry
Size/Mass
Charge
Hydrophobicity
Antibody affinity
Protein Tags
Often used in combination

20. Size Exclusion Chromatography
Separation based on size of protein

21. Ion exchange & hydrophobicity
Non-tagged proteins
Separation based on charge or degree of hydrophobicity
Bound proteins are eluted with salt containing buffers

22. Ion exchange

23. HPLC High performance (pressure) liquid chromatography
Sample is passed over column of varying hydrophobic nature- more hydrophobic particles bind tighter and elutes later.
Eluate is analyzed by a detector
UV, refractive index, fluorescence
Can be combined with mass spec (LC/MS)

24. HPLC

25. Affinity/Ab columns Purify tagged proteins
Interaction between two molecules
Solid phase- immobilized on column
Mobile phase- binds while passing over column
Buffer conditions regulate binding & dissociation
pH, ionic strength, competing

26. Ab affinity column
Include Protein A-agarose/sepharose

27. Tagging the protein
Clone gene in frame with a unique protein sequence or “tag”
Advantages
Purification
Use tag to selectively remove protein from a complex sample
Protein visualization/tracking
Fluorescent protein tags, labeled antibodies

28. Protein Tags His GST S-Tag, C-myc, HA, flag small
6 HIS residues bind to nickel columns
GST
Binds to glutathione resin/beads
S-Tag, C-myc, HA, flag
Antibody affinity columns

29. Protein Tags
6xHistidine binds metal chelating resin- Cu2+, Ni2+, Co 2+

30. What can you do with purified proteins?
Biochemical and functional characterization
DNA binding, enzyme activity, stability, etc.,
Structural analyses informs function
NMR, crystallography, circular dichroism
Study protein-protein or protein-DNA interactions
Develop antibodies

31. Structural Analyses Circular dichroism (basic 2o structure)
Nuclear Magnetic Resonance
Protein Data Bank- pdb.org
X-ray Crystallography
Electron Microscopy
Using structure to inform drug design/mutagenesis

33. Using multiple fragment binding in an enzyme active site to determine possible directions of “growth chemistry” within the active site.

34. Structure of the final inhibitor (IC50 = 3.7 nM)
Fragment linking: X-ray crystal structure of fragments binding at different sites of thrombin
S2–S4 sites (IC50 = 12 μM)
S1 site (IC50 = 330 μM)
Structure of the final inhibitor (IC50 = 3.7 nM)

35. Protein-protein interactions
Two hybrid system
Co-immunoprecipitation
Surface plasmon resonance
Protein arrays
Protein crosslinking
FRET- fluorescence Resonance Energy Transfer

36. Immunoprecipitation Specific Ab binds protein in solution
Solution is eluted over Protein A column
Protein is eluted from Ab
Co-IP
Also allows for study of proteins bound to IP’d protein
ID protein complexes

37. Isolation and identification of protein binding partners

38. SPR Surface plasmon resonance Biomolecular interaction analysis
BIACORE
Protein is immobilized onto surface
Light is refracted onto thin metal layers
Immobile protein refractive index changes when ligand is bound

39. Protein arrays ELISA based format: Ab, proteins, peptides immobilized
Solution to be searched is layered on top
Binding of partner proteins is detected by SPR or fluorescence

40. Protein crosslinking X-linking agent “locks” interacting proteins
Formaldehyde
Linking is highly specific
Can be performed in vivo
Cell extract can be subjected to IP assays
Identify x-linked proteins via co-IP
Chromatin IP – ID bound DNA sequences

41. Fluorescent protein tags
Protein-protein interactions with fluorescence energy transfer (FRET)
Visualizing protein localization
Green fluorescent protein (GFP)