Formal Report - Subcellular Components

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT

SUBCELLULAR COMPONENTS Agustin, Ara Vanessa; Asierto, Faye Julienne; Bondoc, Jao; Bongolan, Abigail; Nicolas, Jomari; Simsuangco, Joanna Faye; Totto, Nikki Mae MT213

Group 5

Date Performed: January 17, 2017

Date Submitted: January 31, 2017

Institute of Arts and Sciences, Far Eastern University, Recto Ave., 395, Manila, 1008 ABSTRACT The cell is known to be the basic unit of life or often called the “building blocks of life" which is compose of different subcellular components that have different biological molecules. All cells have DNA, which is the hereditary material of genes, and RNA that contains the information necessary to build various proteins such as enzymes. The objectives of this experiment are to extract cell organelles through homogenization and to separate the different cell organelles through centrifugation specifically through differential centrifugation. In addition to that, this experiment will give the identification of the presence of biomolecules using different qualitative test. For the laboratory set-up, prepare a fresh chicken liver and blend it. Transfer some on a centrifuge tube and continue with the differential centrifugation. After that, proceed with the qualitative test; different reagents are needed to complete the experiment like NSS, Molisch reagent, Orcinol reagent and etc. Therefore at the end of this experiment, you will learn about the principles of separation of subcellular components. For the expected results on the qualitative test, the color would turn into color blue for the positive result of DNA, blue to blue-green for RNA, purple/pink for proteins, soluble red-orange for lipids and purple for carbohydrates.

1.0 INTRODUCTION Cell is the basic living unit of all

specific functions inside the cell are called organelles.

organisms. It is very small but contains all the characteristics of life. It performs several

To be able to study cell and its functions,

functions such as cell metabolism and energy

it is important to know what components make up

use, synthesis of molecules, communication,

the organelles of the cell. Biomolecules are

reproduction and inheritance (VanPutte et al,

molecules that are involved in the maintenance

2016). The specialized structures that perform

and metabolic processes of living organisms.

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These molecules namely, nucleic acids, proteins,

the tube. To accelerate this process, the lysate

lipids

the

can be subjected to centrifugation. In this

organelles. The isolation of pure components of

process, the lysate is rotated at a certain speed

the cell is essential for these molecules to be

expressed as rotations per minute (RPM). This

studied.

rotation imposes a force on the particles

and

carbohydrates

constitute

perpendicular to the axis of rotation. The force is Subcellular fractionation is the process

called a relative centrifugal force (RCF),

of producing pure fractions of cell components.

expressed as a multiple of the force of Earth's

This method is used when studying subcellular

gravitational force. When a particle is subjected

components for it isolates different organelles

to centrifugal force, it will migrate away from the

from one another in order to be subjected to

axis of rotation at a rate dependent on the

further tests and analysis.

particle's size and density.

The process involves two basic steps:

The specific centrifugation scheme used

disruption of the tissue and lysis of the cells,

in this experiment is differential centrifugation. It

followed by centrifugation, (Gallik, 2011). Figure 1

is the sequential centrifugation of a cell lysate at

at Appendix A shows the steps on subcellular

progressively increasing centrifugation force,

fractionation. The first one’s objective is to

isolating cellular components of decreasing size

disaggregate the cells and break them open with

and density.

The separation of the cellular

minimum damage to the cellular fraction of

components

is

interest. This can be performed in three different

sedimentation rate through the centrifugation

methods (1.) Homogenization breaks up the

medium, which is dependent on the size and

tissue apart and lysed the cells through

shape of the cellular components. Figure 3 of

mechanical homogenizer, such as blender. (2.)

Appendix A shows centrifuged cell homogenate

Sonication uses ultrasound to disrupt the cells. It

that are subjected to different speeds.

based

solely

on

their

is often used when prokaryotic cells are to be lysed. (3.) Osmotic lysis deals with the

This experiment focuses on separating

vulnerability of cells to osmotic stresses. Red

subcellular components through the use of

blood cells can easily be lysed through osmotic

subcellular fractionation. It aims to determine

stress. The process of breaking tissues and cells

what biological molecules are present on each

is shown in Figure 2 of the Appendix A.

fraction separated and analyze how frequent is their appearance on each fraction.

Most of the subcellular components in a lysed cell will eventually settle at the bottom of

2.0 METHODOLOGY

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After two hours, 250 mL of NSS was added again 2.1 Materials and Reagents

to the chicken liver and used blender to emulsify

Different laboratory apparatuses and

it. The chicken liver is then homogenized at

reagents were used to identify the biomolecules

liquefy speed for 10 minutes. Each group got

present in the different fractions namely, Pyrex

their samples in vials and subjected it to

test tubes, vials, 10 ml graduated cylinder,

differential centrifugation. By using centrifugation,

centrifuge, blender, pipette, droppers, beaker,

it can easily separate the various, since the

iron stand, Bunsen burner, and water bath.

various organelles are of different mass, and

Reagents like Normal saline solution and distilled

density. During centrifugation, different organelles

water was used to prepare the chicken liver for

will form pellet at the bottom at specific speeds

homogenization. Concentrated Sulfuric acid and

based on the mass and densities of the

diphenylamine reagent was used in Dische

organelles. The sample was subjected to the first

reaction, a general test for DNA. For the Orcinol

centrifugation at 2rpm for 5 minutes. One

test, which is a test for RNA, freshly prepared

member of the group decants the supernatant

Orcinol reagent was used. Biuret test for proteins

from the centrifugation to another vial. The group

was done by the used of 10% NaOH, 0.5%

then obtained precipitate 1 and filtrate 1. For the

CuSO4 solution, and 1% albumin solution for

second centrifugation, filtrate 1 was put in the

control test. In the test for lipids, chloroform and

centrifuge and spin at 5 rpm for 15 minutes. The

Sudan

for

supernatant was decant to another vial as the

carbohydrates was obtained by the used of

filtrate 2, and so precipitate 2 was attained. For

Molisch reagent, and concentrated sulfuric acid.

the third and last centrifugation, filtrate 2 was

Figure 1 at Appendix B shows the different

subjected to centrifugation at 8rpm for 20

reagents used in the experiment.

minutes. The filtrate 3 was discarded and got the

IV

was

used.

Molisch

test

precipitate 3. The group collected precipitate 1, 2 2.2 Cell fractionation Cell fractionation is used to break open the cells and separate the various organelles. To

and 3 in different test tubes for further study. For the cell suspension 10 ml of NSS was added to each precipitate.

perform cell fractionation, the cells need to be suspended first in solution, and then break open the cell. Normal saline solution was used to wash

2.3 Qualitative test 2. 3. 1 Nucleic acids

60 grams of chicken liver to irrigate tissues for the collection of the organelles. In addition to it,

2. 3. 1. 1 DNA

chicken liver was soaked in distilled water for hemolysis. This will release the organelles inside.

To 6 drops of cell suspension, 4 drops of diphenylamine was added followed by 2 drop

BIOCHEMISTRY (CHEM 24) LABORATORY FORMAL REPORT

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of sulfuric acid in a test tube. The mixture was

2. 4 Observation and Recording

then heated in a water bath for 2 minutes. The

The positive and negative results of bio-

procedure was done 3 times with different cell

molecules in each solution were observed in the

suspensions.

basis of the results that was given by the

2. 3. 1. 2 RNA

professor as shown in Table (1). Table

To 6 drops of cell suspension, 4 drop of freshly prepared Orcinol reagent in a test tube.

Biological Molecules DNA

bath for 2 minutes. The procedure was done 3 times with different cell suspensions.

In the experiment with proteins also known as Biuret Test, 6 drops of cell suspension in a test

Positive

results

for

the

Qualitative tests of Subcellular Components.

The solution was then heated in a boiling water

2. 3. 2 Proteins

1.

Positive Result Blue, Crumpled DNA Fibers Blue to Green,

RNA Protein Lipids Carbohydrates

Crumpled RNA Fibers Purple or Pink Soluble Red-Orange Purple

3.0 RESULTS AND DISCUSSIONS

tube, 6 drops of biuret reagent was added which

This experiment aimed to identify the

was composed of 2 drops 10% NaOH and 0.5%

principles

CuSO4 soln. 1% albumin soln. was used as a

components, the principles in using differential

control test. The procedure was done 3 times

configuration to isolate subcellular components

with different cell suspensions.

and to the biomolecules present in different

2. 3. 3 Lipids

4 drops of chloroform, followed by a small amount of Sudan IV. 2. 3. 4 Carbohydrates

separation

of

subcellular

fractions using qualitative tests.

In the experiment with lipids, a 2 ml cell suspension placed in a test tube was mixed with

of

The cell is composed of cell organelles that have different biomolecule compositions. Cell is the smallest unit of life that can replicate independently, and cells are often called the building blocks of life.

In the experiment with carbohydrates also known as Molisch Test, 2ml of cell suspension in

The experiment was conducted using

a test tube was mixed with 4 drops of Molisch

three general procedures: Extraction, process of

reagent. 2ml of concentrated Sulfuric acid was

isolating the organelles, Homogenization, a

then carefully added and the color of the ring was

process to disrupt the cell and Differential

observed and noted.

Centrifugation, a process to separate the cell organelles. Figure 2 at Appendix B shows the

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homogenized

the

Dische test gave a negative result because it had

experiment. Normal Saline Solution was the main

a cloudy appearance in the solution while Orcinol

reagent used to sterilize the chicken liver. This

test also gave a negative result since the color

reagent was used so as to prevent the

produced after doing this test was light brown

dehydration of the cell. There were different

solution. In precipitate II, both tests gave a

processes made to have three filtrates and three

negative result because in Dische reaction it also

precipitate,

the

had a cloudy appearance in the solution while

I,

Orcinol test had a light brown solution. Lastly, in

qualitative

liver

which tests

sample

were, that

used

needed

were

in

for

Precipitate

Precipitate II and Precipitate III.

precipitate III both were negative because the Dische test had a cloudy appearance and the

The three precipitates were used in

Orcinol test had a color of cream. In addition to

different qualitative tests which are: Nucleic acid

the color changes here in Nucleic Acids, one of

particularly DNA and RNA, Proteins (Biuret test),

the indication if the tests are positive is when you

Lipids and Carbohydrates (Molisch Test).

have a crumpled fibers in the solution. In the experiment, crumpled fibers were there in the

Dische Diphenylamine Reaction is used to detect the presence of DNA in a substance. In

solution and that means DNA and RNA were present.

this process, the reaction between the Dische reagent and 2-deoxypentose results in the development of a blue color. The reaction depends on the conversion of the pentose to whydroxylaevulinic aldehyde, which then reacts with diphenylamine to give a blue colored

Figure 1. The reaction with diphenylamine

complex. The intensity of the blue color is

caused the reaction to create a blue-colored

proportional to the concentration of DNA. This

complex.

test is used to detect the presence of RNA in a substance. This reaction will produce a bluegreen color. The students made several tests and they yielded a cream color and it had a white

Figure 2. The reaction of Orcinol reagent,

precipitate and that means this test gave a

which gives of the blue or blue-green

negative result because there was no RNA

complex.

present in the solution. Dische reagent does not react with the ribose sugar in RNA and does not

Biuret Test this test is used to detect the

form a blue-colored complex. In precipitate I,

presence of a dipeptide. In the presence of

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peptides, a copper (II) ion forms violet-colored

Unfortunately, precipitate I gave a negative result

coordination complexes in an alkaline solution.

because there was no purple color at the

The students made several tests using the given

junction.

solutions. The three precipitates gave positive results in doing this test because the solution was purple and that means there was a protein present in the solutions.

Figure 3. The reaction of biuret reagent

Figure 4. The reactions in the Molisch test are

causing it to violet color.

not specific for a certain carbohydrate but a negative result shows good evidence of their

Sudan IV is classified as an oxidizing

absence. Further test are required if a positive

dye, carcinogen. It is used to stain lipids and

(red or purple) result is seen.

triglycerides. Sudan IV is nonpolar, thus, it only dissolves in nonpolar substances. Again, the

The following results for precipitates I, II

three precipitate gave a positive result because

and III are shown in figures 1, 2 and 3 of

all of them turned into reddish-orange right after

Appendix C.

the small crystals of Sudan IV added. Students cannot avoid making mistakes Molisch test is a general test for

while performing an experiment. It is the

carbohydrates. Molisch is composed of 1%

students’ job to study well the procedures in the

alphanapthol in ethanol with addition of

experiments for them to be able to perform the

concentrated H2SO4. The reagent’s reaction to

experiment well. Accuracy is a must. The

furfural or hydroxymethylfurfural formed by

possible sources of errors could be, the

adding sulphuric acid will cause the presence of

containers used in the experiment are not that

a violet ring. In precipitate II and III, this test gave

clean or maybe there are still residues remaining

positive results because it had a purple ring at

in there that is why some of the tests will give a

the

were

negative result. It might be the case that the

solution.

reagents provided by the laboratory are

junction,

carbohydrates

which present

means in

there the

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contaminated with other solution. It also might be

pestle; third is centrifugation which is done by

because of the concentration of biomolecules for

putting the solution of homogenized organelles in

a certain tests are not enough to show a positive

a variable speed centrifuge and rotating them at

result. Most of the qualitative tests were negative

a higher rate of speed. In general, nuclei are the

in RNA because RNA is a messy folder full of

heaviest subcellular component. The next is

notes that can be scattered around the cell. Also

composed of the mitochondria and lastly the

it might be the case that the solutions given are

supernatant is composed of the least dense

already expired and the laboratory technicians

components

did not notice it at all. Sometimes, it is all about

endoplasmic reticulum, vesicles, ribosomes and

the students who were not able to perform the

cell membrane. The nucleus gave a positive

procedures in their experiment that is why when

result for the biuret test, which indicates the

they perform their tasks they will get a negative

presence of proteins. A positive result for Sudan

result. This only shows that it is important to read

IV test indicates the presence of lipids. The

or understand well the experiment. In the

mitochondria

experiment, the chicken liver might not blend

molecules,

properly which might be able to be one source of

proteins, lipids and nucleic acid. The supernatant

error. Also, it is suggested to heat the solution for

gave a positive result for the biuret test, Molisch

faster reaction like in DNA and RNA tests. Some

test and Sudan IV indicating the presence of

of the qualitative tests were negative because of

carbohydrates, proteins and lipids. The bigger

errors in the procedure.

and/or heavier molecules are found in the

such

as

contain which

are

Golgi

the the

four

apparatus,

biological

carbohydrates,

sediment, while the lighter components are on 4.0 CONCLUSIONS AND RECOMMENDATION

the supernatant liquid.

In this experiment, the students were able to identify the principles used in differential centrifugation

for

isolating

In order to get positive result students

subcellular

should have a proper knowledge about the

components and the biological molecules that

experiment to be performed. Careful execution of

are present in the different fractions using

the procedures could help in achieving the

qualitative tests. The three general procedures in

positive results. Also, the reagents from the

this experiment were extraction which is the first

laboratory should be prepared well in order to

step toward isolating any subcellular component;

prevent errors in the experiment. Another factor

second is homogenization which involves the

is to increase the concentration of the subcellular

breaking apart of cells; thus, releasing the

components to be able to attain an indication of

organelles and cytoplasm. This can also be done

positive results.

by thawing mechanical means using mortar and

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5.0 REFERENCES

Cinnamon VanPutte, J. R. (2016). Seeley's

Alcantara, R. (2014, 11 30). Prezi. Retrieved 01

Essentials of Anatomy and Physiology . McGraw

28,

Hill Education .

2017,

from

https://prezi.com/c1dpzhcczkxz/subcellularcomponent-experiment-2/

Stephen Gallik, P. D. (2011). Cell Biology OLM.

Biochemistry, T. C. (2014). Laboratory Manual

Retrieved 01 28, 2017, from

and Guide Notes in Biochemistry . Brightkids Publication .

http://cellbiologyolm.stevegallik.org/node /74

6.0 APPENDIX 6.1 Appendix A (Subcellular Fractionation)

Figure 1. Subcellular Fractionation

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Figure 2. Tissue/ Cell Homogenization

Figure 3. Differential Centrifugation

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6.2 Appendix B (Procedure of the Experiment)

Figure 1. Reagents Used in the Experiment

Figure 2. Homogenized Liver

6.3 Appendix C (Qualitative Tests Results)

Figure 1. Results in Precipitate I (Nuclear Fraction)

Figure 2. Results in Precipitate II (Mitochondrial Fraction)

Figure 3. Results in Precipitate III (Mircosomal Fraction)