Biochem34.1 Exp2 Report

7/29/2019 Biochem34.1 Exp2 Report

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Biochem34.1Experiment 2Differential Centrifugation of

Subcellular Componentsand Qualitative Analysis ofBiomolecules


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Introduction

Eukaryotic cells are the most studied cellsbecause of their highly organized andcomplex structures.

To be able to study these structures, aprocess called cell fractionation wasdone.

Here, cells are broken open, and the

cellular components are separated onthe basis of size, mass, and density using avariety of centrifugation techniques.


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Introduction Scientists could then isolate and analyze

cell components of different densities,called fractions.

Using this method, biologists had divided

the cell into four fractions: nuclei,mitochondrial-rich fraction, microsomes,and cytosol.

As biochemists, we are interested in the

biomolecules present and theirconcentration levels in these fractionsnamely: nucleic acids, proteins,carbohydrates and lipids.


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ExperimentalLiver

Pellet

+ Buffer

(A)

+ Triton X

-100 (B)

Supernate

Pellet

+ Buffer

(C)

+ Triton X

-

100 (D)

Supernate

+ Buffer

(E)

+ Triton X

-

100 (F)

For Exp. 3

For Exp. 6

6000 rpm,

20 mins

2000 rpm,

10 mins


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Experimental

MOLISCH TEST

Experimental : Cell Suspension + Molischreagent(1% alpha naphthol in alcohol)

+conc H2SO4 Control : 1% glucose + Molisch

reagent(1% alpha naphthol in alcohol)+conc H2SO4

Blank : distilled H2O + Molischreagent(1%alpha naphthol in alcohol) +conc H2SO4


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Experimental

BIURET TEST

Experimental : Cell Suspension +

10%NaOH + 0.5% CuSO4

Control : 1% albumin + 10% NaOH

+ 0.5%CuSO4

Blank : distilled H2O + 10% NaOH +

0.5%CuSO4


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ExperimentalSUDAN IV TEST:

Experimental : Cell Suspension + SudanIV

Control : 1% oleic acid + Sudan IV

Blank : distilled H2O + Sudan IV

SCHIFFS TEST

Experimental : Cell Suspension + Schiffsreagent

Control : 1% oleic acid + Schiffs reagent

Blank : distilled H2O + Schiffs reagent


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Results

Sample Appearance

Pellet 1 Course-grained, containedwhite granules, brown in

color

Pellet 2 Fine-grained, brown in colorSupernate Dark red in color, thick liquid

consistency, has yellowishpowder formation on top of

the liquid


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Results

For the Qualitative Tests, thefollowing results implies positive

results: Biuret: violet

Schiff: pink

Sudan IV: red orange

Molisch: presence of purple ring


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ResultsTest Positi

veBlan

kA B C D E F

Molisch

+++ - +++ + +++ + +++ +

Biuret

+++ - ++ + + + + -

Suda

n IV

+++ - - +++ - + + +


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Discussion


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Discussion Sub-cellular fractionation is done to be able

to further study specific organelles of thecells which cannot be supplied bymicroscopy.

To be able to do this, one must be able tophysically breach the cells by literallybreaking the cells apart.

In our experiment, we used a homogenizing

blender to break up the liver tissues alongwith phosphate buffer with pH 7.5


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Why add buffers?We are trying to simulate the same

environment where our cell samples camefrom.

Remember how a lot of biomolecules arevery sensitive to environment change, weare preventing the alterations theenvironment may do to our cells in that canaffect the tests.

Cells are usually found in an environmentwith a narrow pH range around 7. This

justifies the buffer used with a pH of 7.5which is within the pH range of 8-9.


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Discussion In the experiment, differential centrifugation is

done which utilizes the different densities of

the organelles.

Nuclei already sediment at low accelerationsthat can be achieved with bench-top

centrifuges. Decanting the residue (thesupernatant) and carefully suspending the

sediment (or pellet) in an isotonicmedium

yields a fraction that is enriched with nuclei.

However, this fraction may still contain othercellular components as contaminantse. g.,

fragments of the cytoskeleton.


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Discussion Particles that are smaller and less dense than

the nuclei can be obtained by step-by step

acceleration of the gravity on the supernatant

left over from the first centrifugation.

However, this requires very powerfulcentrifuges (high-speed centrifuges and

ultracentrifuges). The sequence in which the

fractions are obtained is: mitochondria,

membrane vesicles, and ribosomes(microsomes). Finally, the supernatant from

the last centrifugation contains the cytosol

with the cells soluble components, in addition

to the buffer.


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DiscussionThe isolation steps are carried out at

low temperatures on principle (usually05 C), to slow down degradation

reactionse. g., due to releasedenzymes and other influencing factors.

The addition of thiols and chelatingagents protects functional SH groups

from oxidation. Isolated cell organelles quickly lose

their biological activity despite theseprecautions.


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Why add Triton X-100? Triton X-100 is a non-denaturing detergent

that aids in penetrating the cell membranewithout denaturing the cell organelles.

Detergents enable further solubility ofbiomolecules in the buffer.

The addition of detergent increases theconcentration or amount of organelles inthe pellet.

It also aids in studying organelles bound inprotein membranes.


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1. How does denaturation

affect sedimentation ofmacromolecules?

Generally, the denaturation of

proteins makes them aggregate

and become heavier. They

sediment at lower centrifugal

forces. So, once denaturation

occurs, the use of differentialcentrifugation is not likely to

succeed.


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2. What is the purpose ofhomogenizing your sample?

Homogenization disrupts the cell

membrane which encases the

organelles inside a cell and is

involved in the cellular processes

such as adhesion, ion channel

conductance and cell signaling.


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3. What conclusions can be

obtained from the results ofyour qualitative tests?

The presence of lipids and

nucleic acid may indicatewhat organelle is present fromthe pellet produced at acertain centrifuge speed


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4. What other techniques may beemployed to separate subcellularcomponents?

Aside from differential

centrifugation, we could also

employ the density gradient

centrifugation since density is one

of the bases for separation of the

components of the cells.


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Sources: http://stainsfile.info/StainsFile/stain/schiff/reaction-nucleal.htm http://www.harpercollege.edu/tm-

ps/chm/100/dgodambe/thedisk/carbo/molisch/molisch.htm http://www.swccd.edu/~mstinson/biology%20100/biology%20100%

20index/Bio100/Struct-Function%20Cell%20Orga.pdf Murray, Robert K., et al. (2003) Harper's Illustrated Biochemistry. New

York: McGraw-Hill Companies, Inc. Metzler, David E. (2001) Biochemistry:The Chemical Reactions

of Living Cells. Massachusetts: Academic Press Cox, Michael M. and Nelson, David L. (2004) Lehninger Principles Of

Biochemistry, Fourth Edition. New York: W. H. Freeman Koolman, Jan and Rohm, Klaus-Heinrich (2005) Color Atlas of

Biochemistry 2nd Edition. New York: Thieme Stuttgart

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Biochem34.1 Exp2 Report