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Protocols > Thylakoid extraction

Thylakoid isolation method for analysis of protein complexes with Native-PAGE

P1 BUFFER for grinding (1000 ml)

Add before using:

P2 BUFFER for shock (500 ml)

HEPES pH 7.5		50 mM (25 ml of 1 M HEPES-KOH pH 7.5)
MgCl₂		5 mM (2.5 ml of 1 M stock)

Add before using:

NaF*

10 mM (M=42 g/mol)

P3 BUFFER for storage (500 ml)

Add before using:

NaF*

10 mM (M=42 g/mol)

*Please note that NaF is toxic!

For a short-term storage, keep the buffers in 4°C. For long-term storage, keep buffers in -20°C.

Cut the leaves, grind immediately in ice cold P1 buffer (at least 80 ml per sample) in a cold room in dim light conditions.
Filter through miracloth.
Spin at 4°C for 7000 rpm for 5 min.
Re-suspend the pellet in P2 buffer (5 ml for 1 tube, combine the tubes and add 5 ml more of P2 buffer).
Spin at 4°C for 7000 rpm for 5 min.
Discard the supernatant carefully and re-suspend the pellet into a small volume (150-500 µl) of P3 buffer (pipet, paintbrush, vortex, glass homogenizer).
Divide the thylakoids into small aliquots (1 tube for chlorophyll determination, 4 for other analysis) and snap freeze in liquid nitrogen, store at -80°C.

(Porra et al. 1978)

Blank sample - 1000 µl of 80% acetone.
Prepare the sample in triplicates: 5 µl of thylakoids + 995 µl of 80% acetone.
Centrifuge for 5-10 min at 12 000 x g.
Measure the absorbance at 3 different wavelengths 646.6 nm, 663.6 nm and 750 nm.
Use an Excell sheet to determine the chlorophyll content.

Method according to: Järvi, S., Suorsa, M., Paakkarinen, V., and Aro, E.-M. (2011). Optimized native gel systems for separation of thylakoid protein complexes: Novel super- and mega-complexes.