Basic Proteomics for Protein Quantitation and SDS Page

this slide contain about the basical technique for proteomic approach, such as how to measure protein quantity and SDS Page to characterize protein

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 R 2 recommended minimal at 0.99

23. Measuring Protein Quantity Use a Known Concentration Serial Standard to Valid Estimate your Protein Concentration R 2 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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