Biology Reference
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temperature for 30 min in darkness. Measure the absorbance at
595 nm of each sample and standards. Store the protein
extracts at −20 °C for further analysis.
The TCA/acetone-phenol protocol provided the best results in
terms of spot focusing, resolved spots, spot intensity, unique spots
detected, and reproducibility [ 13 ]. We propose to use it to extract
proteins from leaves and pollen [ 16 , 31 ], although these procedures
can also be applied to plant proteomic analysis in general.
3.3 Protein
Extraction from Pollen
by the TCA/Acetone-
Phenol Method
1. The pollen (100 mg) is transferred into a 2 mL tube with 1 mL
of a solution of 10 % (w/v) TCA/acetone. Mix well using a
micropestle and then by vortexing ( see Notes 6 and 8 ).
2. Sonicate 3× 10 s (50 W, amplitude 60) at 4 °C.
3. Fill the tube with the solution of 10 % (w/v) TCA/acetone.
Mix well by vortexing and centrifuge at 16,000 × g at 4 °C for
5 min. Remove the supernatant by decanting.
4. Fill the tube with 0.1 M ammonium acetate in 80 % (v/v)
methanol. Mix well by vortexing and centrifuge at 16,000 × g
at 4 °C for 5 min. Discard the supernatant.
5. Fill the tube with a solution of 80 % (v/v) acetone. Mix well by
vortexing and centrifuge at 16,000 × g at 4 °C for 5 min.
Discard the supernatant.
6. Air-dry the pellet at room temperature to remove residual ace-
tone ( see Note 9 ).
7. Add 1.2 mL of 1:1 phenol (pH 8, Sigma)/SDS buffer. Mix
well using a pipette and by vortexing. Incubate for 5 min on
ice and centrifuge at 16,000 × g for 5 min. Transfer the upper
phenol phase into a new 1.5-mL tube ( see Note 11 ).
8. Fill the tube with a solution of 0.1 M ammonium acetate in
100 % (v/v) methanol, mix well and complete the precipita-
tion overnight at −20 °C.
9. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the
supernatant (a white pellet should be visible).
10. Wash the pellet with 100 % (v/v) methanol and mix by vortex-
ing. Centrifuge at 16,000 × g at 4 °C for 5 min and discard the
supernatant.
11. Wash the pellet with 80 % (v/v) acetone and mix by vortexing.
Centrifuge at 16,000 × g at 4 °C for 5 min and discard the
supernatant.
12. Dry the pellet at room temperature.
13. Dissolve the proteins in the solubilization solution for 2 h,
shaking in a microtube mixer at 4 °C ( see Note 10 ).
14. Procedures for quantify proteins are the same as described in
Subheading 3.2 ( steps 7 and 8 ). Store the protein extracts at
−20 °C for further analysis.
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