Glycerol gradient sedimentation and trichloroacetic acid precipitation of proteins

Glycerol Gradient Sedimentation and Trichloroacetic Acid Precipitation of Proteins

Reagents/Materials

• Glycerol (Sigma-Aldrich, G5516)
• NaOH (Sigma-Aldrich, 655104)
• HCl 1.0 N (Sigma-Aldrich H9892)
• HEPES pH 7.9 (Sigma-Aldrich H4034)
• KCl (VWR chemicals, 26760-295 )
• EDTA (Sigma-Aldrich E1644)
• Nonidet P40 (Sigma-Aldrich 74385)
• DTT (Sigma-Aldrich, D9779)
• Centrifugation tubes: 13.2 mL, Thinwall Polypropylene Tubes, 14 x 89mm – 50Pk (Beckman Coulter, 331372)
• 1.5 mL tubes: Eppendorf® Safe-Lock microcentrifuge tubes (Sigma-Aldrich, T9661)
• cOmplete™, EDTA-Free protease inhibitor cocktail (Sigma-Aldrich, 11873580001)
• Trichloroacetic acid (Sigma-Aldrich T0699)
• Acetone (Sigma-Aldrich 32201)
• Tris base (Sigma-Aldrich T1503)
• SDS (Sigma-Aldrich L3771)
• Bromophenol blue (Sigma-Aldrich, 114391)

Equipment

• Micro centrifuge
• Weighting balance
• Glass beaker
• pH meter
• Measuring cylinders
• Pipettes
• Optima XE-90 Ultracentrifuge (Beckman Coulter)
• SW 41 Ti Swinging-Bucket rotor (Beckman Coulter 331362)
• Disposable transfer pipette (Corning™ Sterile Polyethylene Transfer Pipettes)

Steps

1. Prepare solutions and buffers 
   1. Stock solutions: prepare solutions by dissolving the appropriate amount of powder reagent in distilled H₂O or measuring volume per volume for solutions (Nonidet P40, Glycerol, Acetone) using glass beakers. Note: To adjust pH of solutions HEPES pH 7.9 and Tris pH 6.8 you can use either HCl or NaOH to acidify or alkalify the solution with the aid of  pH meter. Add base or acid drop wise until the desired pH is reached.

                2. Buffers

1. Buffer A: 20 mm HEPES, pH 7.9, 0.3 mM KCl, 0.2 mM EDTA, 0.1% Nonidet P-40, 2mM DTT
2. Buffer A + PI: 20 mm HEPES, pH 7.9, 0.3 mM KCl, 0.2 mM EDTA, 0.1% Nonidet P-40, 2mM DTT, 1x EDTA-free protease inhibitor cocktail (dissolving 1 tablet in 1mL dH₂O yields 50x stock)
3. 4x Laemmli buffer: 0.250 M Tris pH 6.8, 8% SDS, 40% Glycerol, 200 mg Bromophenol blue

b. Glycerol gradient sedimentation (adapted from Contreras X et al., J Biol. Chem., 2009)

1. Prepare glycerol fractions: 10, 15, 20, 25 and 30% v/v in buffer A.
2. Pipette 2 ml of the first fraction (30%) into polypropylene centrifugation tubes. To create the gradient the fractions can be pipetted slowly one by one, or each fraction can be snap frozen in liquid N₂ or at -80°C and a new fraction can be added on top. 
3. Leave gradients overnight (or >6 hours) at 4^oC to form.
4. Lyse cells in 1 mL of buffer A + PI  for 30 minutes on ice.
5. Centrifuge lysates at 20000 xg for 10 min at 4°C.
6. Load supernatants onto glycerol gradients, equilibrate weight of the tubes with Buffer A as necessary to achieve exactly the same weight on opposite sides of the rotor.
7. Fractionate lysates by centrifugation in a SW 41 Ti Swinging-Bucket rotor at 38 000 rpm (for this model; 250000 xg) for 20 hours.
8. Collect 10 fractions (1ml each) from the top in 1.5mL tubes and proceed to protein precipitation.

c. Trichloroacetic acid (TCA) precipitation of proteins (adapted from Link AJ and LaBaer J, Cold Spring Harb Protoc, 2011)

1. Add 0.11 volumes of ice-cold 100% TCA to the protein sample (i.e. if your protein sample is 1mL, you can add 0.11 mL).
2. Place on ice for 10 min.
3. Add 500 μL of ice-cold 10% TCA to the sample.
4. Place on ice for 20 min.
5. Centrifuge at 20,000 xg for 30 min.
6. Carefully remove the supernatant. Aspirate the supernatant with a pipette or a disposable transfer pipette. Avoid disturbing the protein pellet.
7. Add 500 μL of 100% acetone. Very gently rock the tube once or twice to rinse the tube and pellet.
8. Centrifuge at 20,000xg for 10 min. Very carefully remove the supernatant to avoid disturbing the protein pellet. Aspirate the supernatant with a pipette or a disposable transfer pipette.
9. Dry the protein pellet by letting the acetone evaporate at room temperature with the lid of the tube open.
10. Add 50-100 µL of 1x Laemmli Buffer. 
    1. Note: Laemmli buffer is blue because of the bromophenol blue (blue at pH > 4.6), but when the solution is acidic it turns yellow (pH < 3) (reversible reaction). This will be a great indicator if the acetone has been fully evaporated or not. If when adding Laemmli buffer the solution turns yellow, it is important to adjust the pH with a base. 
11. Boil sample at 95°C for 5 minutes. 
12. Sample is ready for SDS-PAGE electrophoresis.