Sample preparation methods

Cell culture supernatant

• Pipette cell culture media into a centrifuge tube and centrifuge at 1,500 rpm for 10 min at 4°C.
• Immediately aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.

Cell extract

• Place tissue culture plates on ice.
• Aspirate medium and gently wash cells once with ice-cold PBS.
• Aspirate PBS and add 0.5 mL complete extraction buffer per 100 mm plate.
• Scrape cells to collect in tilted plate and remove to pre-chilled tube.
• Vortex briefly and incubate on ice for 15-30 min.
• Centrifuge at 13,000 rpm for 10 min at 4°C to pellet insoluble contents.
• Aliquot supernatant (this is the soluble cell extract) to clean, chilled tubes on ice and store samples at -80°C. Minimize freeze/thaw cycles.

Conditioned medium

• Place cells in complete (serum-containing) growth medium and allow cells to proliferate to desired level of confluence.
• Remove growth medium and wash very gently with a few mL of warm PBS. Repeat wash step.
• Remove last PBS wash and gently add serum free growth medium.
• Incubate 1-2 days.
• Pipette medium into a centrifuge tube and centrifuge at 1,500 rpm for 10 min at 4°C.
• Immediately aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.

Milk

• Collect samples and centrifuge at 10,000 x g for 2 min at 4°C.
• Aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.

Plasma

• Collect whole blood into anti-coagulant containing tube, such as BD Vacutainer Coagulation tubes (Cat #: 363080/363080) or add 0.1 M sodium citrate to 1/10 final volume.
• Centrifuge at 3,000 rpm for 10 min at 4°C.
• Immediately aliquot supernatant (plasma) and store samples at -80°C. Minimize freeze/thaw cycles.

Urine

• Collect samples and centrifuge at 10,000 x g for 2 min at 4°C.
• Aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.

Saliva

• Collect samples and centrifuge at 10,000 x g for 2 min at 4°C. 
• Aliquot supernatant and store samples at -80°C. Minimize freeze/thaw cycles.

Serum

• Collect whole blood in untreated test tube or, for example, an anti-coagulant free tube such as BD Vacutainer Serum tubes (Cat #: 367812).
• Incubate undisturbed at room temperature for 20 min.
• Centrifuge at 3,000 rpm for 10 min at 4°C.
• Immediately aliquot supernatant (serum) and store samples at -80°C. Minimize freeze/thaw cycles.

Tissue extract

• Dissect the tissue of interest with clean tools, on ice preferably and as quickly as possible to prevent degradation by proteases.
• Place the tissue in round bottom microfuge tubes and immerse in liquid nitrogen to “snap freeze”. Store samples at -80°C for later use or keep on ice for immediate homogenization.
• For a ~5 mg piece of tissue, add ~300 µL complete extraction buffer (see cell/tissue extraction buffer recipe) to the tube and homogenize with an electric homogenizer.
• Rinse the blade twice using 300 µL complete extraction buffer for each rinse, then maintain constant agitation for 2 hr at 4°C (e.g. place on an orbital shaker in the cold room).
• Centrifuge for 20 min at 13,000 rpm at 4°C. Place on ice, aliquot supernatant (this is the soluble protein extract) to a fresh, chilled tube and store samples at -80°C. Minimize freeze/thaw cycles.

Volumes of lysis buffer must be determined in relation to the amount of tissue present. Typical concentration of final protein extract is >1 mg/mL.