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Protein extract from cells and have the investigation, Summaries of Biochemistry

Protein extract from the cells to how the proteins are present in the cell and have the discussion on that

Typology: Summaries

2022/2023

Uploaded on 07/07/2023

mamatha-singh
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Hands-on workshop on Cancer Tools and
Techniques
April 2023
1
Protein extraction and SDS-Page
Merck Innovation Lab, Bangalore
Uchenna W, Ph.D.
26th April 2023
Protein-Introduction
Structure & Function
Sample preparation
SDS-PAGE
Application
Contents
https://www.uihere.com/
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Techniques

Protein extraction and SDS-Page Merck Innovation Lab, Bangalore Uchenna W, Ph.D. 26 th^ April 2023

 Protein-Introduction

 Structure & Function

 Sample preparation

 SDS-PAGE

 Application

Contents https://www.uihere.com/

Techniques

https://www.intechopen.com/ Expression

Genetic information determines

how are we build, look & function

Proteins Genome - The Instruction Manual of Life Protein: Key Molecule

 Important class of Biological Macromolecules

 Polymers of amino acids, joined by Peptide bonds

 They do most of the work in cells, provide energy

 Required for the structure, function, and regulation

 20 different amino acids Peptide bond

Techniques

Sickle Cell Disease

One Mutation- Cause the disease

https://link.springer.com/article/10.1007/s00277-019-03813-9 https://www.aboutkidshealth.ca/Article?contentid=745&language=English Levels of Protein Structure

 Primary Structure

 Secondary Structure

 Tertiary Structure

 Quaternary Structure

https://pdb101.rcsb.org/global-health/diabetes-mellitus/drugs/insulin/insulin

Techniques

https://commons.wikimedia.org/wiki/File:Protein_primary_structure.svg https://www.quora.com/What-are-the-levels-of-a-protein-structure Protein Primary Structure Protein Secondary Structure

-helix -sheet

 Spatial arrangement of amino acid residues (local folded structures)

 Hydrogen bonds between peptides (intermolecular and intramolecular)

Image credit: OpenStax Biology

Techniques

Levels of Protein Structures

Alphabets Words Sentences Paragraphs

https://alevelnotes.com/notes/biology/biological-molecules/biological-molecules/protein-structure Types of Interactions

Techniques

1 2 3 4 Therapeutics Diagnosis^ Biomarkers^ R & D Protein based drugs Antibodies Insulin Cytokines Teriparatide Hormones levels Renal disease Cancer Diabetes Protein Biomarkers PSA Her- Amyloid beta α-synuclein Protein-protein interaction Protein function Protein structure Importance of studying proteins Protein Extraction https://www.sonomechanics.com/cell-disruption/ https://www.thermofisher.com/

Techniques

Loading

buffer

SDS

DTT

ß-ME

BPB

Glycerol

Tracking dye Migrate with the leading edge of the proteins being separated Increases density of sample Allow the samples to sink into each well Reduce disulfide ridges between cysteines Denatures, linearizes the proteins, coat them with negative charge Loading buffer Protein Separation

Sodium Dodecyl Sulfate

Polyacrylamide Gel

Electrophoresis

Techniques

SDS-Polyacrylamide Gel Reagent Function Acrylamide Forms linear polymers Bis-acrylamide create crosslinks between acrylamide Tris HCl Buffer (7-9 pH range) 10% SDS Denature the protein 10% APS oxidizing agent (produce free radicals) TEMED Catalyze the polymerization β-ME Reduces the di-sulfide bonds

Components

Pre-casted Gel

Separation of proteins from a sample (lysate)

Requirements

Techniques

_

Running Buffer
  • Tris-Base, pH 8.
  • Glycine
  • SDS

SDS-PAGE Electrophoresis

Protein first
Stack
then
Separate
Based on their
size

Thank you