Dasar-dasar Kimia Hayati (KI-1213) (bagian 2) F. Warganegara 7/2/2014KI

Topik Bahasan  Struktur protein  Protein sebagai biokatalis 5. Teknik- Teknik Analisis Sel:  Struktur sel dalam mikroskop  Isolasi dan penumbuh- an sel  Fraksinasi dan Analisis Komponen Sel  Sel dalam Bioteknologi modern 7/2/2014KI Pengantar : 2. Tipe dan Jenis Sel:  Prokarya ke Eukarya  Uni - Multi-selular 3. Molekul, Energi dan Metabolisme:  Komponen Kimia Sel  Energi dan keteraturan Biologi 4. Struktur, Bentuk dan Informasi makromolekul  struktur dan fungsi asam Nukleat

Teknik- Teknik Analisis Sel 7/2/2014KI

5 Struktur Sel di bawah Mikroskop  Mikroskopi: ◦ Magnification, Contrast, Resolution ◦ Light Microscopy (200 nm)  Brightfield  Fluorescent  Advanced ◦ Electron Microscopy (1 nm)  Transmission  Scanning Copyright (c) by W. H. Freeman and Company

5.1 Microskop cahaya Copyright (c) by W. H. Freeman and Company Figure 5-2

5.1.1 Brightfield microscopy  Masalah: Sel umumnya tak berwarna & transparan ◦ Agar strutkturnya terlihat  perlu diwarnai ◦ Dapat melekat dan mudah dibuat irisan yang sangat tipis  Masalah baru yang timbul akibat tindakan di atas: ◦ Mengubah struktur sel/molekul ◦ Hanya memberikan gambaran “sel mati” Copyright (c) by W. H. Freeman and Company

5.1.2 Fluorescent microscopy  Memungkinkan lokalisasi molekul sel spesifik  Pewarna Fluorescent “berpendar” di dalam gelap  Zat warna tsb dapat langsung atau tidak langsung berasosiasi dengan molekul sel  Beberapa pewarna fluoresen dapat digunakan secara berturutan  Dengan mikroskop ini dapat ditampilkan sel mati atau hidup Copyright (c) by W. H. Freeman and Company

Bagan alat Mikroskop fluoresen Figure 5-5 Figure 5-6

Mikroskopi cahaya untuk objek 3-D Mikroskopi cahaya untuk objek 3-D Copyright (c) by W. H. Freeman and Company Figure 5-9 z Confocal Scanning or Deconvolution Microscopy y Generates 3D images of living cells y Removes out-of-focus images  optical sectioning y Can look inside thick specimens (eggs, embryos, tissues)

5.1.3 Advanced light microscopy  Memungkinkan pengamatan sel hidup yang transparan  Pergeseran fasa cahaya (Light phase shifts) yang disebabkan oleh specimen digunakan untuk menciptakan contrast ◦ Phase contrast (refracted and unrefracted light) ◦ Differential interference contrast (two light beams) Copyright (c) by W. H. Freeman and Company Figure 5-14

5.2 Mikroskop Elektron Transmission electron microscopy (TEM)  Operates in vacuum  Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material Special techniques:  Metal shadowing: visualize surface structures, cell components  Cryoelectron: visualize unfixed, unstained samples  Freeze fracture, freeze etch: visualize membrane interior  Freeze etch: visualize cell interior Copyright (c) by W. H. Freeman and Company

Bagan alat Transmission electron microscope Copyright (c) by W. H. Freeman and Company Figure 5-16Figure 5-15

5.2.2 Scanning electron microscopy  Can visualize surfaces of tissues, cells, isolated cell parts  Specimen is fixed and coated with thin layer of heavy metal  Images secondary electrons, resolution = 10 nm Copyright (c) by W. H. Freeman and Company Figure 5-20

5.3 Pemurnian sel dari campurannya dengan flow cytometry Copyright (c) by W. H. Freeman and Company Figure 5-21 Membutuhkan penanda yang berfluorescent untuk sel target

5.2 Pemurnian bagian-bagian sel/organel  Understanding the roles of each each cell component depends on methods to break open (lyse) cells and separate cell components for analysis  Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution  Separation of cell components generally involves centrifugation Copyright (c) by W. H. Freeman and Company

5.3.2 Faraksinasi sel dengan sentrifugasi differensial Copyright (c) by W. H. Freeman and Company Figure 5-23

5.3.3 Pemisahan Organel dengan sentrifugasi kesetimbangan gradien densitas Copyright (c) by W. H. Freeman and Company Figure 5-24

5.4 Struktur sel hewan Copyright (c) by W. H. Freeman and Company Figure 5-42

5.4 Struktur sel tanaman Copyright (c) by W. H. Freeman and Company Figure 5-43

5.4 Organel-organel sel eukaryot  Lysosomes  Peroxisomes  Mitochondria  Chloroplasts  the Endoplasmic Reticulum (ER)  the Golgi complex  the Nucleus  the Cytosol Copyright (c) by W. H. Freeman and Company

5.4 Lysosomes  Responsible for degrading  certain cell components  material internalized from the extracellular environment  Key Features ◦ single membrane ◦ pH of lumen  5 ◦ acid hydrolases carry out degradation reactions Copyright (c) by W. H. Freeman and Company Figure 5-44a

5.4 Peroxisomes  Responsible for degrading  fatty acids  toxic compounds  Key Features ◦ single membrane ◦ contain oxidases and catalase Copyright (c) by W. H. Freeman and Company

5.4 Mitochondria  Site of ATP production via aerobic metabolism  Key Features ◦ outer membrane ◦ intermembrane space ◦ inner membrane ◦ matrix Copyright (c) by W. H. Freeman and Company Figure 5-45

5.4 Chloroplasts  Site of photosynthesis in plants and green algae  Key Features ◦ outer membrane ◦ intermembrane space ◦ inner membrane ◦ stroma ◦ thylakoid membrane ◦ thylakoid lumen Copyright (c) by W. H. Freeman and Company Figure 5-46

5.4 The endoplasmic reticulum (ER)  Responsible for  most lipid synthesis  most membrane protein synthesis  Ca ++ ion storage  detoxification  Key Features ◦ network of interconnected closed membrane tubules and vesicles ◦ composed of smooth and rough regions Copyright (c) by W. H. Freeman and Company Figure 5-47

5.4 The Golgi complex  Modifies and sorts most ER products  Key Features ◦ series of flattened compartments & vesicles ◦ composed of 3 regions: cis (entry), medial, trans (exit) ◦ each region contains different set of modifying enzymes Copyright (c) by W. H. Freeman and Company Figure 5-49

5.4 The nucleus  Separates ◦ DNA from cytosol ◦ transcription from translation  Key Features ◦ outer membrane ◦ inner membrane ◦ nuclear pores ◦ nucleolus Copyright (c) by W. H. Freeman and Company Figure 5-50

5.4 The cytosol  The portion of the cell enclosed by the plasma membrane but not part of any organelle  Key Features ◦ the cytoskeleton ◦ polyribosomes ◦ metabolic enzymes Copyright (c) by W. H. Freeman and Company Figure 5-52