Western blot_生物行

Thursday, December 04, 2003

Description
After extraction of soluble protein from leaves, samples are run on a SDS-PAGE gel. The method according to Sambrook et al. (1989) is used for SDS-PAGE and the method as described in the technical booklet for the ECL Plus Western Blotting system (Amersham, UK) is used for Western Blotting.

Procedure
After electrophoresis, the PAGE gel is equilibrated for 20 min in transfer buffer (25mM Tris/ 192mM Glycine in 15% methanol, pH 8.2). A polyvinylidene difluoride (PVDF) membrane (PVDF-Plus, Micron Separations Inc., USA) is cut to size and pre-wet in 100% methanol for 5 seconds, after which the membrane is washed in distilled water for 5 min, and equilibrated in transfer buffer (500 mL) for 15 min. Transfer of proteins is done in a BioRad Mini protein II transfer apparatus after Sambrook et al. (1989) filled with transfer buffer, at 4篊 and 60V for 1 h. After transfer, the membrane is washed once for 5 minutes in Tris buffered saline (TBS) pH 7.6. Thereafter the membrane is incubated in a 5% fat free milk powder/TBS buffer solution containing 0.1% (v/v) Tween20 for one hour. The membrane is then washed three times for 10 min each in the same milk powder/TBS/Tween solution, and once for 10 min in TBS containing 0.1% Tween20 only (TTBS).

The membrane is incubated in primary antibody in TTBS containing 5% fat free milk powder at room temperature, on an orbital shaker, overnight. To remove all unbound antibodies, the membrane is washed four times for 10 min each in TTBS. Detection is done using the ECLPlus Western blotting detection system (Amersham Pharmacia Biotech, UK), as outlined by the supplier. The membrane is incubated in secondary antibody (HRP labelled goat anti-rat antibody) diluted in TTBS containing 5% fat free milk powder for 2 hours. Afterwards, the membrane is washed four times for 10 min each in TTBS. Detection reagent (2mL) containing a Lumigen PS-3 acridan substrate is pipetted onto the membrane, and incubated for 5 min at room temperature. Excess detection reagent is drained off by touching the edge of the membrane against a tissue. The membrane is wrapped in clean Saran Wrap, and placed in an x-ray film cassette. A sheet of autoradiography film (Hyperfilm ECL, Amersham Pharmacia Biotech) is placed on top of the membrane in a dark room under red safe light conditions. After exposure, the film is developed, rinsed in water, and fixed.

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Western blot