Assay for Proteasome-Dependent Protein Degradation and Ubiquitinated Proteins - Plant Proteomics: Methods and Protocols - page 629

Fig. 2 Agro-infi ltration into tobacco leaf. Agrobacterium suspension with 1 mL needle-less syringe is injected
into abaxial side of the tobacco leaf. The Agrobacterium contains vectors X having genes coding FLAG-tagged
ubiquitin ligase (ATL31-FLAG), Myc-tagged target protein (Myc-14-3-3 ), GFP as control, and p19 protein.

All the constructed genes are constitutively expressed under the CaMV 35S promoter

χ

4. Remove the supernatant and resuspend the pellet with 1 mL
infi ltration buffer ( see Note 5 ) supplemented with 1 μ L

M).
5. Mix the Agrobacterium suspension each with the equal
volume.
6. Inject the Agrobacterium suspension (around 500

Acetosyringone (Final 150 μ

L/leaf)
into the tobacco leaf with a needle-less syringe and incubate
for 2-4 days ( see Fig. 2 , Notes 6 and 7 ).

μ

1. Excise the infi ltrated tobacco leaf and grind it with liquid
nitrogen.
2. Add the 500

3.1.2 Protein Extraction
and MG132 Treatment

L protein extraction buffer and transfer to the
1.5 mL tube on ice.
3. Mix and split the extraction into two different tubes and add
the MG132 (fi nal 30

μ

M) to one tube and DMSO to the
other. Incubate the samples at room temperature for 2 h.
4. Quantify the total amount of protein in the extraction before
proceeding further.
5. Stop the degradation reaction by adding an equal volume of
2× SDS sample buffer.

μ

1. Heat the sample for 5 min at 75 °C and centrifuge by 20,000 × g
for 5 min.
2. Collect the supernatant and for use with SDS-PAGE
( see Note 8 ).
3. Load the 3

3.1.3 SDS-PAGE
and Western Blotting

g protein and start the electrophoresis.
4. Stop the electrophoresis and transfer the protein onto the
PVDF membrane.

μ