The document outlines the fundamental techniques for protein extraction and characterization in proteomics, emphasizing the importance of effective sample preparation and compatible analytical methods. It details various techniques such as RIPA buffer usage, centrifugation, salting in/out methods, dialysis, and various assays like BCA and Bradford for measuring protein concentration. Additionally, electrophoresis techniques like SDS-PAGE are discussed for the separation and characterization of proteins based on their size and charge.

1. Proteomics Basic World:
Characterization and
Identification
Leonardo Tejo Gunawan,S.Si.,M.Biotek
Hanyang Biomedical Research Institute
Lab Division of StemCell Biology and Regenerative Medicine
2024.05.18

2. Introduction
Proteins are fundamental to virtually
every biological process. Identifying
and understanding proteins is crucial
for unraveling cellular mechanisms.
Understanding techniques in basic
Proteomic (at least) can have
BIG IMPACT

3. Introduction
We’ll explore the journey
of protein analysis, from
extraction to
understanding their
functions.

4. “OMICS” research trend over the years

5. Basic Proteomics :
Extraction and
Measuring Protein
01

6. Sample Preparation

7. Remember This!
•Keep the methods as simple as you can —minimize the number of steps and
avoid difficult manipulations which will not reproduce.
•Keep it cheap—avoid expensive techniques where a cheaper one will do.
•Adopt a step approach—and optimize each step as you go.
•Speed is important—avoid delays and slow equipment.
•Use reliable techniques and apparatus.
•Spend money on simple bits and pieces—e.g. test-tubes, pipettes.
•Write out your methods before you start and record what you have done
accurately.

8. Make Your Own Version

9. Or Just Follow the Guideline from Manufacturer

10. Extraction Time!!
Protein extraction is a critical step in studying proteins, as it allows us to isolate them from cells or
tissues.
The efficiency of this process and its compatibility with downstream applications are key factors in
choosing the right extraction method

11. Extraction Time!!
Protein extraction is a critical step in studying proteins, as it allows us to isolate them from cells or
tissues. The efficiency of this process and its compatibility with downstream applications are key
factors in choosing the right extraction method
(Mostly, But Not Limited)
When choosing a protein extraction method, consider your downstream applications.
The method must be compatible with your analytical techniques and should preserve the integrity
and functionality of the proteins or protein complexes you're studying

12. RIPA Lysis Buffer
RIPA buffer, short for Radio-Immunoprecipitation Assay buffer, is a versatile lysis
solution. It contains a mixture of detergents like SDS and/or Triton X-100, along with
sodium deoxycholate, salts, and buffering agents. This combination makes RIPA
highly effective at solubilizing proteins from various cellular compartments, including
membranes, nuclei, and the cytoplasm

13. Another Step / Technique in Protein
Extraction
CENTRIFUGATION
is a technique that separates components in a mixture based on their
density by spinning samples at high speeds. There are two main
types: differential centrifugation, which separates particles based on
size and density, and density gradient centrifugation, which
separates them based on buoyant density.

14. Another Step / Technique in Protein
Extraction
Precipitation
involves altering the solubility of
proteins to cause them to come out
of solution. This can be achieved by
adding salts like ammonium sulfate
or solvents like trichloroacetic acid.
Precipitation is useful for
concentrating proteins and
removing impurities

15. Salting In / Salting Out Method
Salting In refers to the phenomenon where the solubility of a protein increases with the
addition of low concentrations of salt. The added salt ions shield the electrostatic
interactions between protein molecules, preventing aggregation and increasing
solubility. Common salts used for salting in include sodium chloride and potassium
chloride

16. Salting In / Salting Out Method
Salting Out is the opposite process, where the solubility of a protein decreases with the
addition of high concentrations of salt. At high salt concentrations, salt ions compete
with proteins for water molecules, leading to protein precipitation. Ammonium sulfate
and sodium sulfate are commonly used agents for salting out

17. Another Step / Technique in Protein
Extraction
Dyalisis
uses a semipermeable membrane to separate small molecules from
larger protein molecules by diffusion. This technique is effective for
removing salts, solvents, and other small molecules, and is often
used for buffer exchange and desalting protein solutions after
precipitation or other purification steps

18. Another Step / Technique in Protein
Extraction
REMEMBER!! MWCO (Molecular Weight Cut Off) of
membrane must be smaller than the protein target!!

19. Measuring Protein Quantity
BCA ASSAY is a colorimetric assay used to determine protein concentration in a
sample. It relies on the reduction of copper ions (Cu^2+) to cuprous
ions (Cu^1+) by proteins, which are then detected using bicinchoninic
acid
The principle of the BCA assay involves
two main steps. First, the biuret reaction
occurs, where proteins reduce Cu^2+ to
Cu^1+ in an alkaline environment. Then,
bicinchoninic acid (BCA) chelates the
Cu^1+ ions, forming a purple-colored
complex. This complex has a strong
absorbance at 562 nm, which can be
measured using a spectrophotometer to
determine protein concentration

20. Measuring Protein Quantity
Bradford
ASSAY
The principle of the Bradford assay involves the binding of Coomassie
Brilliant Blue G-250 dye to proteins. In an acidic environment, the dye
binds to proteins primarily through arginine and other basic and
aromatic amino acids, causing the dye to shift from brown to blue.
This color change is measured at an absorbance of 595 nm
The Bradford assay procedure is
straightforward. First, prepare the
Bradford reagent, which is
commercially available and ready to
use. Next, add the protein sample to
the Bradford reagent and mix well.
Incubate the mixture at room
temperature for 5-10 minutes to allow
the color development.

21. 280nm Assay
Measuring protein concentration at 280 nm is a straightforward method based on the UV
absorbance of aromatic amino acids, primarily tryptophan and tyrosine, and to a lesser
extent, cystine. These amino acids absorb UV light strongly at this wavelength.
Beer-Lambert Law: A = εlc
ε (molar extinction coefficient): Specific for each protein
Absorbance directly proportional to protein concentration
However, there are limitations to this method. The
absorption at 280 nm varies depending on the
protein’s amino acid composition, particularly its
tryptophan and tyrosine content. Interference from
nucleic acids and other UV-absorbing contaminants
can affect accuracy. Additionally, this method is not
very accurate for low protein concentrations due to the
low sensitivity
Measuring Protein Quantity

22. Measuring Protein Quantity
Use a Known Concentration Serial Standard to Valid Estimate
your Protein Concentration
R2 recommended minimal at 0.99

23. Measuring Protein Quantity
Use a Known Concentration Serial Standard to Valid Estimate
your Protein Concentration
R2 recommended minimal at 0.99

24. Protein Basic
Characterization
02

25. Electrophoresis
Electrophoresis is a technique used to separate charged molecules, such as DNA, RNA,
and proteins, based on their movement in an electric field. The speed and direction of their
movement depend on the molecule's size, charge, and the medium through which they
move.
•Charged molecules migrate towards opposite charge
•Smaller molecules move faster than larger ones
•Separation achieved based on size and charge
Types of Electrophoresis
Bullet Points:
Agarose Gel Electrophoresis: DNA and RNA separation
Polyacrylamide Gel Electrophoresis (PAGE): Protein
separation
SDS-PAGE: Denatured proteins by size
Native PAGE: Proteins in their native state

26. SDS-PAGE
(Sodium Dodecyl Sulphate-Polyacrilamide Gel Electrophoresis)
involves using sodium dodecyl sulfate (SDS), a detergent that denatures proteins, giving
them a uniform negative charge. This allows proteins to be separated strictly by size when
an electric field is applied. SDS-PAGE is widely used for protein characterization and
assessing protein purity, but also for DNA fingerprinting test.

27. Not Only SDS, but also need other
component
Break dissulfide bonds

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