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Proteomics

Extraction of Protein from Cerebrospinal Fluid

Extraction of Protein from Cerebrospinal FluidProtein extraction from cerebrospinal fluid requires a welloptimized protocol to get the maximum yeild of proteins for takingit for clinical analysis. The method explained here mainly focuseson the effective method of extracting protein from thecerebrospinal fluid.

Learning Objectives:

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

• Identify the extraction technique of proteins from thecerebrospinal fluid.

• Choose to prepare the buffers required for the extraction.• Infer the steps involved during the experiment.• Troubleshoot at various steps 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.


Take the CSF sample out of -80°C freezer and thaw it gradually by placing on ice.

Aliquot out the required amount of CSF into a fresheppendorf tube.

Cerebrospinal Fluid


Fold a fresh butter paper and tare its weight from thebalance.

Reagents Preparation


Prepare lysis buffer consisting of 4M guanidinethiocyanate, 0.5% lauryl sarcosine, 1mM Dithiotreitol,0.3M NaCl and 0.1M Tris base. This buffer helps inefficient lysis of the cells and release of proteins.



Set the pH of the buffer solution to 7.4 using HCl.



Add 1ml lysis buffer to the tube containing CSF andproceed for homogenization.

CSF Protein Extraction


Homogenize the sample using a tissue homogenizer.Carry out the step 4 times by placing the sample in icewith each round lasting for 30 seconds on and 30seconds off.

CSF Homogenization


Incubate the sample at 37°C for 30 minutes followedby incubation at 65°C for another 30 minutes.

Sample Incubation


Centrifuge the contents at 4°C, 14000 rpm for 20minutes. After centrifugation, pellet and supernatantcan be seen clearly.

Sample Centrifugation and Supernatant Collection


Carefully pipette out the supernatant into a fresh tubeand discard the pellet obtained.

Sample Centrifugation and Supernatant Collection


Mix phenol, chloroform and isoamyl alcohol in theratio 25:24:1 for phase separation of proteins, RNAand DNA from the supernatant obtained afterhomogenization.

Biomolecules Separation


Add 350μl of Phenol, chloroform, isoamyl alcoholsolution to the supernatant and vortex the tube for ashort time to mix the contents thoroughly.



Centrifuge the contents at 14000rpm, 4°C for 20minutes to separate the biomolecules in three layers.



Discard the upper layer and the middle layer intofresh appendorf tubes for later use.

The lower phenol layer consists of separated proteins.

Organic Separation


To the tube containing proteins; add 5 volumes ofisoamyl alcohol. Keep the tube undisturbed at roomtemperature for 10 minutes for the proteins toprecipitate.

Protein Precipitation


Centrifuge the tube at 10000rpm, 4°C for 5 minutesfor total precipitation of the proteins.



Discard the supernatant out from the tube and air drythe pellet for 10 minutes.

Once the pellet is air dried completely, dissolve it inrehydration buffer.

The protein dissolved in the rehydration buffer cannow be stored at -20°C for further use.
