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  • Experiment 1 - Subcellular Components

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    Renz Victor Guangco
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    The document describes a process for isolating organelles from cells through differential centrifugation and analyzing the macromolecular components of the organelles. Key steps include homogenizing minced chicken liver in a sucrose solution using an osterizer, then centrifuging the solution at increasing speeds and times to separate out nuclear, mitochondrial, and microsomal fractions based on density. A variety of tests are used to analyze the fractions, including Biuret for proteins, Molisch for carbohydrates, Sudan IV for lipids, Dische reaction for deoxypentose, and Orcinol/Bial for pentose.

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    The document describes a process for isolating organelles from cells through differential centrifugation and analyzing the macromolecular components of the organelles. Key steps include homogenizing minced chicken liver in a sucrose solution using an osterizer, then centrifuging the solution at increasing speeds and times to separate out nuclear, mitochondrial, and microsomal fractions based on density. A variety of tests are used to analyze the fractions, including Biuret for proteins, Molisch for carbohydrates, Sudan IV for lipids, Dische reaction for deoxypentose, and Orcinol/Bial for pentose.

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    4K views2 pages

    Experiment 1 - Subcellular Components

    Uploaded by

    Renz Victor Guangco
    The document describes a process for isolating organelles from cells through differential centrifugation and analyzing the macromolecular components of the organelles. Key steps include homogenizing minced chicken liver in a sucrose solution using an osterizer, then centrifuging the solution at increasing speeds and times to separate out nuclear, mitochondrial, and microsomal fractions based on density. A variety of tests are used to analyze the fractions, including Biuret for proteins, Molisch for carbohydrates, Sudan IV for lipids, Dische reaction for deoxypentose, and Orcinol/Bial for pentose.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
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    of 2

    Isolation of organelles in their pure form and analysis of macromolecular/biomolecular components of the organelles.

    PRINCIPLE INVOLVED: Differential Centrifugation

    ANALYSIS: 1. BIURET TEST - Test for a dipeptide (protein) - Produce pink color from dipeptide to polypeptide. - CuSO4 Oxidizing Agent ; NaOH rxn medium

    STEPS: 1. Extraction - Process of isolating the organelles. - Use Saturated Sucrose Solution (C12H22O11) - Under mild condition of pH and temperature under harsh conditions. (T=4C and <) 2. Homogenization Disrupting the cells under a mild condition using an osterizer. 3. Centrifugation Subfractionalization of the centrifugate.

    2. MOLISCH TEST - General test for carbohydrates. - conc. H2SO4 dehydrates a sugar yielding a furfural. - naphtol in alcohol reacts with furfural to complete a violet/purple colored complex.

    CENTRIFUGATE/RESIDUE: I Nuclear Fraction (Nucleic Acids) II Mitochondrial Fraction (Carbohydrates) III Microsomal Fraction (Lipids)

    3. SUDAN IV - Classified as oxidizing dye, carcinogen. - Hydrophobic dye that dissolves on hydrophobic solvents, lipids. * Non-specific test because all hydrophobic will dissolve in Sudan IV

    4. DISCHE REACTION - For deoxypentose - Diphenylamine (DPA) Putent carcinogen.

    5. ORCINOL/BIAL TEST - For pentose - conc. HCl Dehydrates the pentose to furfural - Orcinol Rgt. Gives color reaction with furfural.

    Minced Chicken Liver + 20mL NSS (Normal Saline Solution) [Put to 5 vacuotainers] 1 Residue (Labeled #1) ; Filtrate 2 Residue (Labeled #2) ; Filtrate 3 Residue (Labeled #3) Filtrate (Discarded) #1: Centrifugated at speed 2 for 10 minutes. #2: Centrifugated at speed 3 for 15 minutes. #3. Centrifugated at speed 4 for 20 minutes. TESTS:
    PRECIPITATE MOLISCH TEST (5 Drops) ORCINOL TEST (2 Drops; Water bath for 5 mins. SUDAN IV (w/ 2 Drops CHCl3/CH2Cl2) DPA (w/ 1 Drop conc. H2SO4 ; Water bath for 5 mins) BIURET ( 2 Drops CuSO4 and NaOH)

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