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Proteomics

Extraction of Brain Tissue Protein

Methodology for the Extraction of Brain Tissue Protein

Extraction of the entire protein from the sample requiresoptimized protocol and many protocols have been developed toincrease the protein amount in the extract from different samples.The method explained here mainly focuses on the effectivemethod of extracting protein from the brain tissue.

Learning Objectives:

After interacting with this learning object, the learner will be able to:

• Define protein extraction of brain tissue using lysis buffer and homogenisation.

• Perform protein solubilisation using rehydration buffer.• Interpret the results of the experiment.• Assess the troubleshooting steps involved in the experiments.

Note: The current IDD exists in two modes- interactive and automatic.Students taking lab course should select interactive (set as default),while the automatic mode may be selected for general users.


Clean the surface of the balance with a tissue paper.Place a butter paper on the balance and tare theweight of the paper.

Weighing the Sample

Weighing the Sample

Take out the tube containing the sample from the -80°C freezer and place it on ice.

Using forceps, pick up the tissue and weigh it on abutter paper.

Transfer the weighed tissue into a fresh appendorftube.


Washing the Tissue

To the tube containing the tissue, add 0.5 ml of PBS.

Vortex the tube for 30 seconds followed by a shortspin for about 10 seconds.

Discard the supernatant.

Repeat the wash step another time.


Tissue Sonication

Now, add 0.5ml of PBS containing protease inhibitorand carry out 6 cycles of sonication, 20% amplitudewith each cycle having 5 seconds on-time and 10seconds off-time.


Tissue Sonication

Centrifuge at 14000 rpm for 5minutes at 4°C.

Carefully transfer out the supernatant obtained into afresh appendorf tube and proceed with Trizoltreatment.


Trizol Treatment

The supernatant obtained is a mixture of all thebiomolecules that make up the cell and predominantlyconsists of proteins, DNA and RNA. TRIzol - chloroformis added to separate proteins from the nucleic acids.


Trizol Treatment

Add 1 ml TRIzol reagent to the supernatant.

Vortex the tube thoroughly to ensure uniform mixingof the supernatant with the reagent.


Chloroform Treatment

Add chloroform to the sample and mix thoroughly tilla milk-shake like solution is formed.

Keep the tube undisturbed for 5 minutes tillseparation of the three phases is seen.


Chloroform Treatment

Centrifuge the tube at 12000rpm, 4 degrees C for10minutes.

A clear separation of the three phases can be seenafter centrifugation is over. The topmost layer consistsof RNA, the middle layer consists of proteins while thelowermost layer is made of DNA.


Absolute Alcohol Treatment

Pipette out the upper aqueous layer containing RNAwithout disturbing the other two layers. Depending onthe need, RNA can either be stored in a new tube orbe discarded.

Now, add absolute alcohol to the tube and mix gentlytill the middle layer dissolves completely. Keep thetube undisturbed at room temperature for 3 minutes.

Centrifuge the tube for 5minutes at 2000rpm.


Acetone Treatment

Upon alcohol addition followed by centrifugation, theDNA forms a pellet in the tube while the proteinsremain a part of the supernatant.

Carefully pipette out the supernatant and transfer itto a fresh tube without disturbing the DNA pellet.

Add chilled acetone to the tube containing thesupernatant and vortex thoroughly to form ahomogenous mix.


Acetone Treatment

Place the tube in a -20 freezer for at least an hour.

After an hour’s incubation remove the tube from thefreezer.

Subject the contents of the tube to centrifugation at12000 rpm for 10minutes and 4 degrees C.

The proteins are seen as a pellet in the tube now.


Wash Buffer Preparation

Clean the surface of the balance. Now place a butterpaper and tare the weight of paper before weighing.


Wash Buffer Preparation

Now weigh the contents for wash solution. Weigh therequired amount of Guanidine Hydrochloride andtransfer it to the tube.

Take the required amount of ethanol in a measuringcylinder and add it to the tube containing guanidinehydrochloride.

Vortex the tube to dissolve the salt and to form auniform solution.

Place the tube in a refrigerator and store it at 4degree C for further use.


Rehydration Buffer Preparation

For making rehydration buffer, weigh 0.02 g of CHAPSand 0.06 g of Urea and dissolve in water.

Add 0.05% of Bromophenol Blue to the rehydrationbuffer. This helps in tracking the sample duringelectrophoretic run.

Add required amount of water and vortex the tube forcomplete mixing of the salts.

Store the tube at 4 degrees C for later use.



Discard the supernatant obtained in the tube withoutdisturbing the protein pellet. Air dry the pellet toremove traces of acetone which could interfere withthe washing step.

Now, proceed with washing to remove the pink colorof the pellet. Add wash buffer to the tube containingthe pellet.

Vortex the contents thoroughly and centrifuge thetube at 12000rpm for 10 minutes.

Again, carefully discard the supernatant withoutdisturbing the pellet and carry out wash step with thepellet till the pink color disappears to white.



To the pellet obtained after washing, add about 0.4 mlof rehydration buffer. Rehydration buffer containsCHAPS which solubilise the proteins, while Ureapresent helps denature the proteins.



Vortex the tube to thoroughly mix the contents.


Sample Storage at -20°C

The sample can be stored at -20°C for use later. Whenneeded for protein quantification, the contents of thetube can be thawed and used.

Please go through the following IDDs for moreinformation.


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