Question

I am trying to isolate the proteins found in the blood of Alzheimer's patients with the hope of finding a biomarker that can potentially indicate the early onset of the disease and was wondering how I could do so? I know centrifugation is an option but how would I go about doing it specifically?

-my end goal is to simply isolate the proteins from the blood samples in order to run ITRAQ analysis on them and MS analysis after

Answer 1

ISOLATION OF PROTEINS FROM BLOOD

• collect the blood sample from the donor and place it on the ice for an hour to allow coagulation of sample.
• To separate the serum from the coagulated blood, centrifuge the sample at 2500 rpm, 4°C for 10 minutes
• upper layered straw coloured serum is collected in a eppendorf tube.
• it can be stored in -80°C for further use.
• 500μl of phosphate buffer is added to the serum sample.
• 8 cycles of sonication is performed at 20% amplitude 5 seconds on and 15 seconds off to bring cell lysis. This step is conducted by placing on the ice.
• Next step is depletion of sample. The depletion is performed for the separation of high molecular weight proteins from low molecular weight protein.
• depletion column is pre treated to room temperature and centrifuge it at 800 rpm for 5 minutes.Add the binding buffer to the column and centrifuge at 2400 rpm for 5 minutes. discard buffer in the column.Now the column is ready.
• To perform depletion add the serum sample to the column and incubate it for 10 minutes on ice . Centrifuge the column at 800 rpm for 30 seconds.
• collect the depleted sample.
• To this sample add 500 μl of 10% TAC-acetone solution and place it in -20°C for 4 hours to precipitate protein.
• sample centrifuge at 14000 rpm, 4°C and 30 minutes.
• at the bottom of the tube proteins are seen as pellets.
• To collect this pellets remove the supernatant by pipetting.
• the pellet is washed with 1ml of ice cold wash buffer.
• Then centrifuge this at 14000 rpm, 4°C for 10 minutes.
• after centrifugation air dry the pellet for 10 minutes and add 400 μl of rehydration buffer to solve the pellet.
• rehydration buffer is prepared using 0.02 gm of CHAPS and 0.6 gm of urea ;the urea denatures the protein and CHAPS solubilizes protein.