Technical information

Antibody types

Purification

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Protocols > TCA acetone precipitation method

TCA/Acetone protein precipitation method for plant proteins.

Procedure

Grind plant tissue in liquid nitrogen.
Continue grinding with 10% TCA solution in acetone (ice-cold).
Precipitate overnight in -20°C.
Spin at 4°C for 1 min, 17000 rpm.
Wash with ice-cold acetone until you obtain a white pellet (usually 2x is enough). Do not let the pellet dry.
Dissolve the pellet in your buffer of choice (for example 8 M urea containing 5 mM DTT, or denature in SDS protein loading buffer for 10 min at 70°C)
Centrifugate briefly, to remove any non dissolved material and use supernatant for further analysis.
Measure protein concentration using commercial reagents compatible with urea and detergents.
Proceed with a Western blot.

Avoid vigorous vortexing and shaking.

Example

This method allows high protein load per well. An example of Western blot results obtained with this method can be found below.

western blot using anti-AGO4 antibodies

360 µg/well of Arabidopsis thaliana protein extracted by TCA-acetone precipitation from floral tissue and saturated in 8 M urea were separated on 15% SDS-PAGE and blotted for 1 h to 0.2 µm nitrocellulose at 100 V using a wet transfer system. Blots were blocked with 0.5% cold fish gelatin for 1 h at RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:2500 for 1 h at RT with agitation. The blots were washed 3 x 15 min with TBS-TT at RT with agitation. Blots as incubated in the secondary antibody (DayLight®800) 1:5000 dilution for 30 min at RT with agitation, and washed once with TBSTT for 15 min, and once with TBST for 15 min before scanning with the ODyssey IRD scanner.

Courtesy of Dr. Betty Chung and Pawel Baster, University of Cambridge, United Kingdom.