Skip to document
Welcome to StudocuSign in to access the best study resources
Guest user
0followers
HomeMy LibraryAsk AI
  • You don't have any recent items yet.
My Library
  • You don't have any courses yet.
  • You don't have any books yet.
  • You don't have any Studylists yet.
Premium
This is a Premium Document. Some documents on Studocu are Premium. Upgrade to Premium to unlock it.

Chicken Liver EXPT Draft

membrane biology notes
Academic year: 2021/2022
Uploaded by:
Anonymous Student
This document has been uploaded by a student, just like you, who decided to remain anonymous.
University of Delhi

Comments

Please sign in or register to post comments.

Students also viewed

Related documents

Preview text

EXPERIMENT 9

AIM: Isolation of Mitochondria from the liver and assay of marker enzyme SDH.

MATERIALS REQUIRED

  • Homogenizer
  • Spectrophotometer
  • Cuvettes
  • Centrifugation tubes
  • Chicken liver

Reagents:

  • 0% saline
  • 0 Tris-Cl Buffer (pH=7) with 0 sucrose
  • 0 M Tris-Cl Buffer( pH=7 )
  • 0 Sodium Azide in 0 Tris-Cl
  • 0 sodium succinate in 0 M Tris- Cl
  • 0 DCPIP

THEORY

Centrifugation is a useful means of isolating and purifying cellular components, because most of them differ from one another significantly in size and density. Because of difference in shape, size and density, the various organelles or other cellular structures can be separated or fractionated. This process is known as sub-cellular fractionation.

Homogenization: It is any of several processes used to make a mixture of two mutually non- soluble liquids the same throughout. This is achieved by turning one of the liquids into a state consisting of extremely small particles distributed uniformly throughout the liquid. Cells which are a part of a solid tissue such as liver or kidney will first need to be separated from all the connections with another cell. In some cases, this can be performed by simply chelating the environment but in most instances the cell will need to be enzymatically or mechanically disaggregated. This often results in subtle changes to the cells .and disrupts such cell-cell communications. The physical means of homogenization is the combined use of mortars and pestles, blenders, compression and/or expansion.

Differential Centrifugation: According to this preparation of broken cells is poured into a centrifuge tube and is initially centrifuged at low centrifugal force long enough to completely sediment the largest and heaviest sub-cellular component. On centrifugation the heaviest particles near the bottom of the tube sediment first while the longer period is required for the same species of the particles which are in the upper region of the centrifuge tube. By the time these get pelleted, lighter particles originally present near the base of the tube also get sedimented within this period. In fact, the pellet obtained is enriched with heaviest particles but is likely to contain other particles also. The lighter particles, freed from sediment and which are towards the top of the tube, will remain in the supernatant. The supernatant obtained is carefully decanted and it is again centrifuged at higher centrifugal force for sedimenting the next heavier entity in the extract. This process is continued and, at each ensuing step, the centrifugal force as well as the centrifugation time is increased to

Sub-cellular fraction or organelle

Marker Plant Cell Animal Cell Nucleus DNA

NMN Adenyl transferase

NADP pyrophosphate

DNA-dependent RNA

Polymerase

DNA

NMN Adenyl transferase

NADP pyrophosphorylase

DNA-dependent RNA

Polymerase I Chloroplast RUBISCO

NADP triose phosphate dehydrogenase

-

Proplastid Ribulose-1,5-

bisphosphate carboxylase

Nitrite reductase

Fatty acid synthase

Cytochrome c oxidase

-

Mitochondrion Cytochrome c oxidase

Succinate dehydrogenase

Succinate: cytochrome c reductase fumarase

Antimycin A insensitive NADH

Cytochrome c reductase

Cytochrome c oxidase

Succinate dehydrogenase

NAD- glutamate dehydrogenase

Peroxisome Catalase

D-amino acid oxidase

rate oxidase

Hydroxypyruvatereductase

Catalase

D-amino acid oxidase

2-hydroxy acid oxidase

rate oxidase Glyoxysome Malate synthetase

Isocitrate lyase

-

REACTION OF DCPIP, REDUCTION TO A

COLOURLESS COMPOUND

  1. Sodium Azide

It will block the electron transfer from FADH2 which instead gives the electrons to DCPIP.

  1. DCPIP

It will act as the electron acceptor in the SDH assay.

ASSAYING SDH IN VITRO

PRINCIPLE

The purpose is to measure the rate of activity of succinate dehydrogenase catalysing the reaction, succinate to fumarate in vitro using a mitochondrial fraction from liver cells.

  • The reaction is measured by observing the reduction of 2,6 dichlorophenolindophenol, and artificial electron acceptor rather than coenzyme Q. Adding Sodium Azide blocks the electron transport system so that electron cannot reduce coenzyme Q. Instead, the electrons are transported from E-FADH2 to DCPIP.
  • The reduction of {DCPIPH2} can be identified by a colour change; the oxidized form of the electron acceptor is blue, and its reduced form is colourless:

E-FADH2 + DCPIP oxd. (blue) ------> E-FAD + DCPIP red. (colourless).

The extent to which DCPIP is oxidized is measured by recording the different absorbances of different enzyme concentrations using a spectrophotometer at 600 nm.

PROCEDURE

Isolation of Mitochondria from Chicken Liver

  1. 5g of chicken liver was washed with 0% saline in a petri dish,

  2. All the contaminants (fats, blood clots, attached tissue) were removed. After that excess of saline was dried with filter paper. With scissors the liver was minced. Then 10 ml of homogenization buffer (0) Tris- Cl buffer pH 7 with 0 M sucrose was added and 100μl of 10% triton X 100 was added and this mixture was transferred to homogenization tube. It was then homogenized in four strokes in a homogenizer (each stroke is about 10-20seconds) with the cooling of homogenate after each stroke.

  3. The crude homogenate obtained was further processed by differential centrifugation. The homogenate was transferred to 2 centrifuge tubes and centrifuged at 1000 rpm for 10 minutes. The pellet (cell debris) was dissolved in 5 ml buffer with sucrose while supernatant was collected and measured.

  4. Out of supernatant, collected 2 ml was kept away in a vial and the rest was again centrifuged at 1000 rpm for 10 minutes. The pellet (cell debris) was dissolved in 5 ml buffer with sucrose while supernatant was collected and measured.

  5. Out of supernatant, collected 2 ml was kept separate in a vial and the rest was again centrifuged at 3000 rpm for 10 min. the pellet obtained was homogenized with a homogenization buffer by a pipette. After homogenization, its volume was measured, and it was kept in a vial. This pellet was the Nuclear Pellet.

  6. The supernatant was again centrifuged at 10,000 rpm for 10 minutes. The pellet obtained was again homogenized with homogenization buffer by a pipette. After homogenization it’s volume was measured and it was kept in a vial. This pellet was Mitochondrial Pellet.

SDH Assay

  1. It is a continuous time assay in which 0 ml of DCPIP mixture containing 0 DCPIP
  2. 1 ml Tris and 0 ml Sodium Azide and 0 ml enzyme fraction were added to cuvette (control set).
  3. Then the cuvette was placed in spectrophotometer to record the drop in OD at 600 nm for a particular fraction, with one-minute interval up to 10 minutes. This gives the endogenous activity.
  4. Then the cuvette was taken out washed and then 0 of DCPIP, 0 ml of enzyme fraction and 0 ml of Sodium succinate were added to cuvette.

RESULT

Activity of different fractions

  1. Crude= 0.
  2. Nuclear=0.
  3. Mitochondrial=0.
  4. Cytosolic=0.

From the result obtained in our experiment the activity of the Mitochondrial fraction was found to be 0, SDH is a marker enzyme for mitochondria ,so it was expected that the SDH activity will be highest in the mitochondrial fraction, which is validated from result obtained.

PRECAUTIONS

  1. Excessive homogenization high temperature should be oxidised because it led to denaturation of protein and it may affect the SDH activity.
  2. Homogenization must be done optimally as excessive homogenization causes disintegration of organelles
  3. Fractions must be kept in ice to prevent enzyme denaturation.

GRAPH FOR Crude and Cytosolic Fraction

GRAPH FOR Mitochondrial and Nuclear fraction

FOR

Was this document helpful?
Premium
This is a Premium Document. Some documents on Studocu are Premium. Upgrade to Premium to unlock it.

Chicken Liver EXPT Draft

Course: Membrane Biology And Bioenergetics (BCH C-6)

University: University of Delhi

Was this document helpful?

This is a preview

Do you want full access? Go Premium and unlock all 10 pages

  • Access to all documents

  • Get Unlimited Downloads

  • Improve your grades

Upload

Share your documents to unlock

Already Premium?
EXPERIMENT 9
AIM: Isolation of Mitochondria from the liver and assay of marker enzyme SDH.
MATERIALS REQUIRED
• Homogenizer
• Spectrophotometer
• Cuvettes
• Centrifugation tubes
• Chicken liver
Reagents:
• 0.9% saline
• 0.15M Tris-Cl Buffer (pH=7.4) with 0.25M sucrose
• 0.4 M Tris-Cl Buffer( pH=7.4 )
• 0.2M Sodium Azide in 0.4M Tris-Cl
• 0.15M sodium succinate in 0.4 M Tris- Cl
• 0.2M DCPIP
THEORY
Centrifugation is a useful means of isolating and purifying cellular components, because most
of them differ from one another significantly in size and density. Because of difference in
shape, size and density, the various organelles or other cellular structures can be separated or
fractionated. This process is known as sub-cellular fractionation.
Homogenization: It is any of several processes used to make a mixture of two mutually non-
soluble liquids the same throughout. This is achieved by turning one of the liquids into a state
consisting of extremely small particles distributed uniformly throughout the liquid. Cells
which are a part of a solid tissue such as liver or kidney will first need to be separated from
all the connections with another cell. In some cases, this can be performed by simply
chelating the environment but in most instances the cell will need to be enzymatically or
mechanically disaggregated. This often results in subtle changes to the cells .and disrupts
such cell-cell communications. The physical means of homogenization is the combined use
of mortars and pestles, blenders, compression and/or expansion.
Differential Centrifugation: According to this preparation of broken cells is poured into a
centrifuge tube and is initially centrifuged at low centrifugal force long enough to completely
sediment the largest and heaviest sub-cellular component. On centrifugation the heaviest
particles near the bottom of the tube sediment first while the longer period is required for the
same species of the particles which are in the upper region of the centrifuge tube. By the time
these get pelleted, lighter particles originally present near the base of the tube also get
sedimented within this period. In fact, the pellet obtained is enriched with heaviest particles
but is likely to contain other particles also. The lighter particles, freed from sediment and
which are towards the top of the tube, will remain in the supernatant. The supernatant
obtained is carefully decanted and it is again centrifuged at higher centrifugal force for
sedimenting the next heavier entity in the extract. This process is continued and, at each
ensuing step, the centrifugal force as well as the centrifugation time is increased to

Why is this page out of focus?

This is a Premium document. Become Premium to read the whole document.

Why is this page out of focus?

This is a Premium document. Become Premium to read the whole document.

Why is this page out of focus?

This is a Premium document. Become Premium to read the whole document.

Students also viewed

Related documents