The extraction of mitochondrial proteins for 2-D electrophoresis

Separation of soluble proteins from membrane bound ones:

  1. Start with about 4 mg of mitochondrial protein (the mitochondria are freshly prepared or stored at -20C).

  2. Resuspend in 1 ml of 20 mM Tris/HCl pH 7.4 buffer using a glass homogenizer and sonicate with a tip sonicator for 1 min (make sure that the sample is not heated!).

  3. Centrifuge for 30 min at 20 000 rpm at 4C.

  4. Separate the pellet (P1) from the supernatant (S1).

  5. Resuspend the pellet (P1) in 1 ml of 20 mM KPi pH 7.4 buffer using a glass homogenizer.

  6. Divide P1 and S1 in two equal portions and precipitate P1 and S1 with 20 TCA according to standard protocol (four samples).

 

Sequential extraction of proteins for IEF:

All solutions should be prepared using the highest grade chemicals. When the components have been dissolved, filter through a 0.2-0.44 µm filter, aliquot and store at -20C.

Tributylphosphine (TBP) should be added to the sample solution only prior the use!

The IPG buffer should be added after solubilisation of the sample!

 

  1. Solubilise P1 and S1 in 350 microl of sample solution A and/or B (four samples).

  2. Add 0.7 microl of TBP (2 mM final concentration).

  3. Incubate for 10-30 min (if it is necessary, even longer) at room temperature (RT).

  4. If it is necessary, sonicate for 1-3 min at RT. Heating also helps speed solubilisation, but it should only be done prior to the addition of urea. It is because heating in the presence of urea can introduce protein charge modifications.

  5. Centrifuge for 10 min at 14 000 rpm at RT and collect the supernatant (S2).

  6. Add 1.8 microl of IPG buffer of appropriate pH gradient (0.5 final concentration) and add a few grains of bromophenol blue.

  7. Apply on the strips (350 microl of sample solution per 18 cm strip) and run the rehydration/isoelectric focusing at 20C and 50 microA/strip:

 

150 V – 1 hr,

 500 V – 1 hr,

 1000 V – 2 hrs,

 4000 V – 4 hrs,

 8000 V – 8 hrs.

In total: 82 650 Vhrs and 16 hrs.

 

Solutions:

Tris T-1378 ‘Sigma’ [20 mM]: 242.2 mg/100 ml H2O, pH 7.4 with HCl

KH2PO4 04873.1000 ‘Merck’ [20 mM]: 272.18 mg/100 ml H2O, pH 7.4

For other solutions see ‘Chemicals and solutions for the IEF…’.

 

2004 02 11 – Ove Julius