Subcellular Fractionation

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The Importance of Subcellular Fractionation

To understand the function of a particular organelle To study metabolic reactions, signaling pathway regulation, processing

and trafficking of molecular components

The very first step is to separate the organelle from other components of the cell (Subcellular Fractionation).

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Purpose of Our Experiment

How? (Experimental design)

Understanding the properties of two organelles

Looking at their differences

To separate the mitochondria and the nucleus from each other and other cellular components

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The Nucleus

Exists only in eukaryotic cells.

Is the largest organelle.

Carries genetic materials. Almost all DNA is in the nucleus.

Contains proteins regulating the expression of genes.

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Mitochondria

Found in most eukaryotic cells.

Convert organic materials into energy in the form of ATP via the process of oxidative phosphorylation.

Enzymes for cellular respiration embedded in membranes.

Contain small circular DNA .

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Questions

Which properties can be used to separate the nucleus and mitochondria?

Which properties can be used to identify the nucleus or mitochondria?

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Property Same or different?

Good or not for separating?

Membrane Same Not Good

Shape Different Not Good

Size Different Method??

Density Different Centrifugation

Content Different Microscopy, Enzymatic Activity

VS

Lyse cells found in Cauliflower

Separate Nuclei and Mitochondria

Identify the Organelles

DNA Staining and Microscopy

Succinate Dehydrogenase

Assay

Differential Centrifugation

Presence of nuclei Presence of mitochondria

Experimental DesignHomogenizati

on

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Part A: Subcellular Fractionation The most common method of separating/organelles is differential centrifugation

The different sedimentation rates/gravity and the density of various cellular components make it possible to separate (pellet) them partially by centrifugation.

Step-wise centrifugation

1000 x g for 5 mins (low speed) denser organelles in pellet, lighter in supernatant

10,000 x g for 30 mins (high speed) sediments/pellets lighter organelles from the supernatant

1. 20 g

2. 40 mL Mannitol Grinding Buffer

4. 1000g x 5 min

5a. Decant supernatant

Nuclei Mitochondria

5b. Add 8 mL Mannitol assay buffer

3a. Filter

Keep everything on ice!!!!!!!

5c. 2 mL

5d. 10,000g x 30 min

Mitochondria

15 mL: Filtrate 15 mL: S1

Centrifuge: P1 Centrifuge: P2

15 mL: S2

15 mL: P1 15 mL: P2

6b - 7. Add 8 mL Mannitol assay buffer

6a. Decant supernatant

3b. 4 mL

Filtrate S1P1

+

1 dropAzure C

How do we verify which fraction contains the nuclei?

1 drop 1 drop 1 drop

+

1 dropAzure C

+

1 dropAzure C

DNA staining and microscopy

Observe unstained slide then add Azure C and observe with stain

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Microscope

Use the microscope guide to setup and analyze of samples

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Nuclei Staining

Nuclei Staining with Azure C

The nuclei are stained blue to purple

Observed as round or oval shaped structures with darker purple round nucleoli within

Mitochondria are too small to observe under light microscopy

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Microscopy: Results of staining fractions with Azure C stain

Fraction Expected results

Observed results

Conclusions

Filtrate ? ? ?

P1 ? ? ?

S1 ? ? ?

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What do we expect to find in each fraction?

Fraction Expected result

Homogenate (cell lysate) Many nucleiMany mitochondria

S1 (supernatant from 1000 xg spin)

Many mitochondria,Few nuclei

P1(pellet from 1000 xg spin)

Many nuclei, Some mitochondria

S2 (supernatant from 10,000 x g spin)

Few mitochondriaNo nuclei

P2 (pellet from 10,000x g spin)

Many mitochondria,Few nuclei

How do we verify the fraction contains mitochondria? We test for the presence of Succinate Dehydrogenase

An enzyme attached to the inner mitochondrion membrane

Involved in TCA cycle and part of complex II of ETC

Catalyzes oxidation of succinate to fumarate

Covalently bound flavin adenine dinucleotide (FAD) is an electron acceptor or coenzyme in aerobic respiration to carry e-s to the ETC

2 e-’s and 2 H+’s removed from succinate and donated to FAD during the Krebs cycle

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SDH

Succinate + FAD Fumarate + FADH2

Succinate Dehydrogenase

ETC

Succinate + FAD Fumarate + FADH2

Succinate Dehydrogenase

2e-

AzideETC

e- accumulation

2e-

https://youtu.be/fBXSJGxfnbU

If the ETC is shut down, e- can not be transferred to final electron acceptor.Limited amount of FAD available, when all FAD has been reduced SDH will use DCIP to continue passing on e-.

Use Dichlorophenolindophenol (DCIP), an artificial electron acceptor, as an color indicator

2e- + 2H+

Succinate

Oxidized form of DCIP (blue)

Fumarate

Reduced form of DCIP

(colorless)

Succinate Deydrogenase

Part B: The SDH Assay - How do we measure SDH activity?

The change in absorbance at 600 nm over time can be used to determine the concentration of DCIP reduced. We will work with different volumes of the fractions to test for the presence of mitochondria by way of testing for the presence of SDH. This will tell us the volume of mitochondria present in a sample.

+ Azide

Part I: Which fraction contains the mitochondria?

Cuvette

Mannitol

Assay Buffer

Azide* (0.04

M)

DCIP(5x10-

4M)

Malonate (0.2M)

Succinate

(0.05M)

Subcellular

fraction sample

 

OD (600nm) at 0 min

OD (600nm) at 3 mins

Filtrate Blank

1.24mL 0.2mL     0.2mL0.36mL Filtrate

0.0 0.0

Filtrate 1.04mL 0.2mL 0.2mL   0.2mL0.36mL Filtrate

   

S1 Blank

1.24mL 0.2mL     0.2mL 0.36mL S1 0.0 0.0

S1 1.04mL 0.2mL 0.2mL   0.2mL 0.36mL S1    P1

Blank1.24mL 0.2mL     0.2mL 0.36mL P1 0.0 0.0

P1 1.04mL 0.2mL 0.2mL   0.2mL 0.36mL P1    P2

Blank1.24mL 0.2mL     0.2mL 0.36mL P2 0.0 0.0

P2 1.04mL 0.2mL 0.2mL   0.2mL 0.36mL P2                     

Malonate

Control0.96mL 0.2mL 0.2mL 0.08mL 0.2mL 0.36mL P2    

Azide control

0.60mL   0.2mL   0.6mL 0.36mL P2    

Succinate

Control1.24mL 0.2mL 0.2mL     0.36mL P2    

Experimental Controls

Controls Effect Subsequent Effect

Expected result

1. Competitive inhibition by malonate

Succinate doesn’t bind

to SDH

2. No sodium azideElectrons continue

down to ETC

3. No succinateSuccinate not available to bind to SDH

Less FADH2 available for reduction of

DCIP

No products (No Fumarate

+ FADH2)

No products(No Fumarate

+ FADH2)

Cannot measure SDH

activityDCIP-Dark blue

No SDH activityDCIP-Dark blue

Very less to No SDH activity

DCIP-Dark blue

Sodium azide- precautions

Wear Nitrile glovesDO NOT touch your face/ exposed skin with the gloves Avoid aerosol formation

ALL WASTE MUST GO IN PROPER WASTE DISPOSAL CONTAINER

Procedure1. Turn on spectrophotometer to warm up for at least 5 mins.

2. Label cuvettes according to the table

3. Add all solutions to each tube EXCEPT the subcellular fraction

4. Cover each cuvette with parafilm and invert twice to mix ***Azide is poisonous

5. Add fraction to BLANKS only, cover and mix

6. For each sample you will be measuring the absorbance at 600 nm at time zero

and at 3 minutes - Timing is critical!!

7. Blank the spec, then add the fraction, mix, start timer and immediately read the

absorbance

8. Blank again BEFORE timer reaches 3 mins and measure absorbance AGAIN at 3

mins – DO NOT mix the sample by inversion

9. Repeat this for each tube

Part II: SDH Assay

Tube

Mannitol

Assay Buffer

Azide* (0.04

M)

DCIP(5x10-

4M)

Succinate

(0.05M)

Subcellular fraction sample

OD (600nm) at 0 min

OD (600nm) at 3 mins

P2 Blank 1.60mL 0.2mL   0.2mL 0.0mL P2 0.0 0.0

P2 1.40mL 0.2mL 0.2mL 0.2mL 0.0mL P2    

P2A Blank

1.48mL 0.2mL   0.2mL 0.12mL P2 0.0 0.0

P2A 1.28mL 0.2mL 0.2mL 0.2mL 0.12mL P2    

P2B Blank

1.36mL 0.2mL   0.2mL 0.24mL P2 0.0 0.0

P2B 1.16mL 0.2mL 0.2mL 0.2mL 0.24mL P2    

P2C Blank

1.24mL 0.2mL   0.2mL 0.36mL P2 0.0 0.0

P2C 1.04mL 0.2mL 0.2mL 0.2mL 0.36mL P2    

S2 control

1.24mL 0.2mL   0.2mL 0.36mL S2 0.0 0.0

S2 1.04mL 0.2mL 0.2mL 0.2mL 0.36mL S2    

Procedure1. Turn on spectrophotometer to warm up for at least 5 mins.

2. Label cuvettes according to the table

3. Add all solutions to each tube EXCEPT the subcellular fraction

4. Cover each cuvette with parafilm and invert twice to mix ***Azide is poisonous

5. Add fraction to BLANKS only, cover and mix

6. For each sample you will be measuring the absorbance at 600 nm at time zero

and at 3 minutes - Timing is critical!!

7. Blank the spec, then add the fraction, mix, start timer and immediately read the

absorbance

8. Blank again BEFORE timer reaches 3 mins and measure absorbance AGAIN at 3

mins – DO NOT mix the sample by inversion

9. Repeat this for each tube

Critical Thinking Questions

1. Are your results consistent with azide functioning as an electron transport chain inhibitor?

2. Is SDH activity dependent upon the presence of mitochondria? Do your results support this?

3. If nuclei are more dense than mitochondria, where would you expect to find nuclei?