Isolation of cellular Proteins.pptx
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This document provides an overview of isolating cellular proteins from bacterial broth. It begins by defining proteins and their important functions in cells. The objectives are to understand proteins, enzymes, and functional proteomics. The experimental work involves isolating total protein from bacterial broth by lysing cells, centrifuging to remove debris, and collecting the supernatant containing soluble proteins. The methodology describes growing and harvesting E. coli cells, washing and lysing them using a lysis buffer, centrifuging to remove debris, and collecting the protein-containing supernatant which can then be stored or used for further analysis. The overall aim is to isolate proteins from E. coli broth culture using cell lysis and centrifugation techniques.
Isolation of cellular Proteins.pptx
- 1. Isolation of cellular Proteins By Okechukwu Francis PhD Biotechnology, UPES
- 2. Objectives of this Overview Understanding protein as a biomolecule Enzymes, Proteins and Functions (Functional Proteomics) Experimental work • Isolation of total protein from bacterial broth References Thank you
- 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 influencing transcription.
- 5. Principle 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 electrophoresis.
- 6. Aim: Isolation of proteins from Escherichia coli broth culture • Materials: • 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 • Microcentrifuge • 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 buffer. 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 cell debris. 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 assays.