2. Objectives of this Overview
Understanding protein as a biomolecule
Enzymes, Proteins and Functions (Functional Proteomics)
• Isolation of total protein from bacterial broth
3. Protein as a biomolecule
Proteins are large, complex molecules made up
of amino acids linked together in a specific
sequence. They perform a diverse range of
functions that are crucial for the structure,
function and regulation of cells and organisms.
4. Functions of Proteins
1. Structural Elements: They form the structural basis of cells, tissues, and organs.
Examples include collagen in connective tissues and keratin in hair and nails.
2. Enzymes: Proteins catalyze chemical reactions in the body, facilitating metabolic
processes essential for life.
3. Hormones: Some proteins, like insulin, act as signaling molecules that regulate
various physiological processes.
4. Transporters: Proteins aid in the transport of molecules across cellular membranes,
facilitating nutrient uptake and waste removal.
5. Antibodies: These proteins are part of the immune system, recognizing and
neutralizing harmful pathogens.
6. Receptors: Proteins on cell surfaces or within cells receive and transmit signals,
regulating processes like growth and development.
7. Motor Proteins: They enable movement within cells, allowing processes like muscle
contraction and intracellular transport.
8. Storage Proteins: Some proteins store essential molecules, such as iron in ferritin.
9. Gene Regulation: Certain proteins control gene expression by binding to DNA and
The protein isolation principle is
based on the combination of steps
which entails that proteins are
cellular components of living cells
and are released on cell lysis. Due
to their density and molecular
structure proteins can be
concentrated by precipitation at a
specific PH and separated based on
their molecular weight by gel
6. Aim: Isolation of proteins from Escherichia coli broth
• E. coli broth culture or Macro Algae broth culture
• Buffers (e.g., Lysis buffer: 10ml Tris-Hcl @PH 7.41, 1ml of 0.5% NP-40, 10ml
of 40mM Nacl, 0.46g EDTA dissolved in 5ml of ddH2O, 0.42g NaF, Top volume
to 200ml and set at PH 7.4)
• PBS buffer
• DNase I
• Protease inhibitors
• Centrifuge tubes (1.5 ml or 15 ml)
• Sonicator or Stomacha
• Refrigerated centrifuge
• Pipettes and tips
• -80C Refrigerator
7. • Methodology:
• Bacterial Culture and Harvest:
1. Grow the E. coli culture in a suitable broth medium to the desired cell density.
2. Harvest 10ml of cells by centrifugation at 4,000rpm for 2 minutes at 4°C.
3. Decant the supernatant, add 5ml of PBS buffer containing protease inhibitors
and vortex for 10 – 30 seconds to wash out excess media.
4. Centrifuge at 4,000rpm for 2 minutes, decant supernatant containing excess
5. Resuspend cell pellet in 1ml of lysis buffer and incubate at room temperature
for 10 minutes to obtain the lysate (you can also place tubes in ultra Sonication
machine for 30 seconds with 1minute intervals for 5 circles if your cell
membrane is more rigid e.g. Algea).
6. Centrifuge the lysate at high speed (4,000rpm) for 2 minutes at 4°C to pellet
7. Carefully transfer the supernatant (containing soluble proteins) to a new tube.
• Storage or Further Analysis:
• Store the protein lysate at -80°C or proceed with downstream applications,
such as SDS PAGE, Western blotting, mass spectrometry, or enzyme activity