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Cellular Response: Biochemical Analysis

Cellular Response: Biochemical Analysis. Dr. M. H. Ghahremani. Reference: 1-Masters, J.R.W. (2000). Animal cell culture, 3rd Ed., Oxford. 2-Sambrook, J.; Russell, D.W. (2001). Molecular cloning: a laboratory manual,3rd Ed., CSH press. The goal of this course. How to get response from cell

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Cellular Response: Biochemical Analysis

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  1. Cellular Response:Biochemical Analysis Dr. M. H. Ghahremani Reference: 1-Masters, J.R.W. (2000). Animal cell culture, 3rd Ed., Oxford. 2-Sambrook, J.; Russell, D.W. (2001). Molecular cloning: a laboratory manual,3rd Ed., CSH press. Dr. M.H.Ghahremani

  2. The goal of this course • How to get response from cell • How to measure changes in cells • What kind of assays one can use in cells • How biochemical assays will provide responses Dr. M.H.Ghahremani

  3. How can we use cell for responses? • Morphological studies • Proliferation studies • Cell Death studies • Biochemical changes • Cell to cell interactions Dr. M.H.Ghahremani

  4. Cellular changes • Transcription changes • mRNA levels • RT-PCR • Real time PCR • Northern blot • Translation changes • Protein levels • Immunohistochemistry • Western blot Dr. M.H.Ghahremani

  5. Biochemical changes In Cytoplasm • Change in second messengers • Change in secretory chemicals • Change in enzymatic activities • Change in protein interactions In nucleus • Change in nuclear enzymes • Change in transcription Dr. M.H.Ghahremani

  6. Second messengers • Cyclic nucleotides • cAMP, cGMP • Interacellular • extracellular • Radioimmunoassay • ELISA • Radioisotope Dr. M.H.Ghahremani

  7. Second messengers • Intracelluar Calcium • Single cell • Cell suspension • Chromophore • Fura2-AM • Radioisotope • Patch clamp Dr. M.H.Ghahremani

  8. Secretory chemicals • Hormones • Neurotransmitters • Signaling molecules • Radioimmunoassay • ELISA • Radioisotope Dr. M.H.Ghahremani

  9. Enzymatic activities • In the intact cell • Extracted form What to consider • Substrate • Reach to enzyme • Quantitative measurement • Low background • Chromophores • Radioisotope Dr. M.H.Ghahremani

  10. Enzymatic activities What to measure • Enzyme kinetic • Enzyme localization • Substrate specificity • Enzymes to measure • PKA, PKC, PLC • MAPK, JNK • COX, LOX • Caspases Dr. M.H.Ghahremani

  11. Phosphorylation • Activation of proteins (or Enz) • Inactivation of proteins (or Enz) How to measure? • Addition or elimination of Phosphate • Using 32P • Using Mass Spectroscopy • Using Specific Antibody • Localization • Substrate specificity • Protein Interactions Dr. M.H.Ghahremani

  12. Protein interactions • Measure physical interaction • Protein-protein interaction • Protein-chemical interaction • Immunopercipitation • Fusion protein • Yeast two hybrid Dr. M.H.Ghahremani

  13. Protein interactions • Measure biochemical interactions • Change in enzymatic activity • Substrate specificity • Effect on other biochemical pathway Dr. M.H.Ghahremani

  14. Protein interactions • Measure Protein-DNA interactions • Change in Interaction • DNA finger printing • Gel shift assay • Yeast One hybrid • Change in Transcription • Effect on other proteins Dr. M.H.Ghahremani

  15. Dr. M.H.Ghahremani

  16. 4.30 Dr. M.H.Ghahremani

  17. Dr. M.H.Ghahremani

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