CBNU Dept. of Animal Science, Graduate School

1. Advanced Cell Biology
2014 1nd Semester
Department of Animal Science
Chungbuk National University
2ndt Lecture

2. 1st week : Introduction
3rd week :Research Strategies For Cell Biology
5nd week : Nucleus, Transcription and Splicing
7nd week : Membrane and Channel
9nd week : Membrane Trafficking
11nd week : Cell Signaling
13nd week : Cytoskeleton
15nd week : Cell Cycle

3. Cell and complicated Machine : The ways to study Cells

4. Component List
What kinds of component are in the machine (or cell?)
But How about the cell?

5. “Component” in the Cell : Protein (RNA)
Genome Sequence : Now we have most of gene list in the genome
But it does not means that we know the exact component list in a specific cells…
http://www.ncbi.nlm.nih.gov
http://genome.ucsc.edu/

6. Differences cells have different component (Proteins, RNA),
although they have common genome
How we can figure out the whole component list?

7. Expression profiling using Microarray or RNA-Seq
“What Kinds of mRNA is there?”
“How much specific RNA is there?”
RT-qPCR or Northern Blot
mRNA levels does not necessarily correlated with Protein Levels.

8. Nature, 2012

9. Proteomics
~less than 1,000 abundant Proteins

11. - “We want to know where the specific component is located inside in the cell”
- “We want to know whether two protein is interact each other in cell”
Immunofluorescence / Flurorescence reporter fusion
Colocalization / Immunoprecipation
- “We want to see what will happen if the specific protein / RNA was devoided in cell”
RNAi
Knockout (CRISPR/Cas9)
Ectopic Expression of Dominant Negative Mutant
Now let’s assume that we have a list of component inside the cell. Now what?
- “We want to check how the shape of the protein looks like”
X-ray Crystallography

12. Immunoprecipitation

14. Optical Microscope : Main workhorse of Cell
Research

15. Phase-Contrast Microscope
Contrast-enhancing techniques

17. Differential-Interference-Contrast Microscope

19. Fluorescences
Fluorescence : some molecules can absorb one color and emits different colors

20. Fluorescences Microscope

22. Filters: the key to successful
fluorescence microscopy

25. Staining of different components of the cell
Q : We want to localize the location of a specific protein in cell. How we can do
that?
A : Use Antibody!
By labeling antibody with fluorescence, you can locate the desired protein in

26. Alpha-Tubulin Actin Mitochondria
Synaptic Vesicle

27. Immunofluorescence
- Direct Immunofluoresence
* Antibody (or chemical) which can bind a desired protein is labeled with
fluorochrome
* Pros
- Convienient
- More Sensitive
* Cons
- You should have a primary antibody labeled
with fluorochrome
- If you don’t have it, you should do it by yourself or
use Indirect Immunofluoresence

28. Alexa-Phalloidin
Phalloidin : Actin binding Chemical

29. - Indirect Immunofluoresence
* Unlabeled Antibody is applied on the fixed tissue
* Antibody was detected by secondary antibody conjugated with fluorochrome
Primary Antibody
Recognize Antigen
Secondary Antibody recognize
Primary Antibody
It is labeled by fluorescence
Pros
• You don’t need to label primary antibody
• Based on the selection of secondary antibody, you can change wavelength of signal
Cons
* More complicated (Two step process)

30. Fixation and Section
We need to stop the cellular process and preserve the component inside in
cell.
Crosslinking Fixation
Commonly used for luoresence microscopy
Generate covalent cross-links between intracellular components
Most commonly used agent : aldehyde
Formation of bond between amine grouop
Glutaraldehyde
formaldehyde
- Precipitating Fixatives :
Disctrupt hydrophobic interaction Denature proteins
Methanol, Ethanol, Acetic Acid

31. Colocalization
Using two different fluorophore with different wavelength, we can test cellular locations of
Two protein simultaneously.
A B

32. Choice of fluorophore
* Choice of two closely distributed spectrum may cause bleeding

34. Fluorescence Protein as Reporter
GFP Gene of Interest
- Drawbacks of immunoflorescences
• You need to have (specific and high-quality) antibody against your protein of interest
• You need to fix a cell (i.e. Dead Cells), so you cannot observe live event in live cell
- You need a probe which will work in In the Living cell (and even organism)
• GFP(RFP) – Your Gene of Interest
• Transfection

35. • Time-Lapse Imaging of Live Cell

36. Confocal Microscopy
• The main problem in the florescence microscopy is that strong
illumination background from other focal planes

38. Biochemical Characterization of Cell

40. Pertubation of Component
• Loss-of-function Study
- Knockdown of functions for GOI (Gene of Interest)
- Find a Phenotype caused by the Ablation of Gene Function
- Find a function of Gene/Protein
- Can be classified as
- RNAi
- Morpholino
- Dominant Negative Mutant
• Gain-In-Function Study
- Introduction / overexpression of GOI
- Find a Phenotype caused by the (over)expression of Gene

41. RNAi (RNA interferences)
• Temporal knockdown of desired gene
• Loss-of function Study

42. Transfection

44. Genome Engineering and Knockout

46. Dominant Negative Mutant
These Proteins are active only if they are exists as dimer..
If we express ‘truncated form’ of mutant, they will be inactivated
regardless of presence of wild type molecule
Endogenous expression

47. Uses of dominant negative Rho family GTPases (Rac, Cdc42, Rho)

48. X-ray Crystallography

49. Protein Productions
- You need to have enough (5-10mg) pure (at least 95% purity) protein
- Overexpression (Bacteria or Insect Cell or Mammalian Cell) or Natural Source
- Purification

50. Crystallization
- Concentrate Proteins (at least 5mg/ml)
- Crystallization happens in the boundary of soluble and precipitation

53. Strong X-ray generated from synchroton is essential

57. Raw Data : Diffraction Images of Protein Crystal
(several hundreds)
Computer
Analysis

58. Final Structure and Interpretations

59. Do I need to know how to solve protein structure?
- Not really (In most cases..)
Do I need to know how to check my favorite protein?
- Yes for sure.
http://www.rcsb.org

60. OK. Let’s check some random guy’s structure 

61. In old days, you need very expensive workstation-level computer
To visualize Protein Structure..
Not anymore. Cheap PC or even your smartphone can do that.

62. Pymol (http://www.pymol.org)
Demo