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Proteomics

Extraction of Proteins from Serum

Methodology for the Extraction of Proteins from SerumExtraction of the entire protein from the complex biological samples likeserum requires a multi -step robust protocol, where various sampleprocessing steps have been introduced to increase efficacy of theextraction procedure.

Learning Objectives:

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

• Define the protein precipitation using TCA-Acetone treatment.

• Infer the protein solubilisation using rehydration buffer.

• Separate out high abundance proteins.

• Operate steps involved in handling the instrument and the material used.

• Assess the troubleshooting steps involved in the experiment.

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 and tare the weightof paper before weighing the reagents.

Reagents Preparation


Prepare 10% TCA-Acetone solution by dissolving 1 gramof trichloroacetic acid and 0.07 grams of Dithiotreitolin 10 ml of acetone.

Dissolve completely by vortexing the tube and store the tube at 4°C for later use.



Weigh 0.02 grams of CHAPS, 0.6 grams of urea anddissolve in water to prepare rehydration buffer.

Add 500μl of bromophenol blue to the tube, vortex itthoroughly and store the tube at 4°C for later use.



For serum sample, collect blood from a donor in aserum separation tube. This should be done verycarefully under the supervision of a skilled person toavoid any accidents.

Blood Processing


Place the tube on ice for about an hour and allowcoagulation of the collected blood sample.

After incubation, centrifuge the tube at 2500 rpm,4°C for 10 minutes to separate serum from thecoagulated blood.

Blood Processing


Collect the upper layer of straw colored serum into afresh eppendorf tube for further processing.

Blood Processing


Make aliquots of serum by distributing into fresheppendorf tubes and store the tubes at -80°C. Thetubes can be used later when required.

Blood Processing


Take out the required amount of serum into a fresheppendorf tube from the serum stock maintained at-80°C.

Place the serum stock tube back in the -80°C freezerafter use.

Sample Processing


Add 500μl of Phosphate buffer to the serum sampletaken in a fresh eppendorf tube.

Sample Processing


Vortex the sample tube to bring about uniform mixingof serum with phosphate buffer. Proceed for cell lysisby sonication once the mixing is complete.

Sample Processing


Sonicate the sample for 8 cycles, 20% amplitude 5seconds on and 15 seconds off. The step is to becarried out by placing on ice since a lot of heat isreleased during sonication.

Sample Processing


Serum contains a large number of high molecularweight proteins like Albumin, α1-antitrypsin,Transferrin, Haptoglobulin, Immunoglobulin G andImmunoglobulin A. These proteins interfere with theseparation of low molecular weight proteins during IEFand hence should be depleted from serum.

Sample Depletion


Take the depletion column out of the refrigerator inorder to ready it for pretreatment.

Sample Depletion


Once the column attains the room temperaturecentrifuge it at 800rpm for 5minutes. This separatesthe storage buffer from the resin.

Sample Depletion


Remove binding buffer from the freezer and add it tothe column.

Centrifuge the column at 2400rpm for 5minutes. Thebinding buffer helps in activating the resin therebycausing better binding.

Sample Depletion


Discard the buffer in the column.

The column is now ready to be used. Add serum to thecolumn and incubate it on ice for 10 minutes.

Sample Depletion


The proteins of high abundance bind to the resin whilethose of low abundance pass through the column. Thedepleted sample is now better suited for separationusing 2D gel electrophoresis.

Depletion Mechanism


Centrifuge the column for 30seconds at 800 rpm. Thishelps in better separation of the high and lowabundance proteins.

TCA-Acetone Precipitation


Collect the depleted sample in a separate tube.

Add 500 μl of 10% TCA-Acetone solution to the tubeand place it in the -20°C freezer for 4 hours toprecipitate the proteins.



Centrifuge the sample at 14000 rpm, 4°C and 30minutes. The proteins would be seen as a pellet at thebottom of the tube.



Separate the supernatant from the pellet by carefulpipetting. Take care not to disturb the pellet.



Add 1ml of ice cold wash buffer for pellet wash.



Disperse the pellet in the wash buffer that has addedby adequate vortexing.



Now centrifuge the tube at 14000rpm, 4°C for 10minutes to pellet the proteins.



Take the tube out of the centrifuge and discard thesupernatant. Air-dry the pellet for 10 minutes and add400μl of rehydration buffer, sufficient to dissolve thepellet.

Vortex the tube to dissolve pellet completely.

Rehydration buffer consists of urea which denaturesthe proteins and CHAPS which solubilizes proteins.

Rehydration Buffer Treatment


Store the sample in -20°C freezer until further use.

Sample Storage