Issue 7
December, 2015

Immunoprecipitation (3)Introduction
Systems such as UPS (ubiquitin-proteasome system) function in the degradation of intracellular proteins, and it is known that many proteins
are involved in a complex manner. So far, proteomics analysis for elucidating the mechanism of UPS has been vigorously carried out, andmany results have been obtained.
The proteasome is a huge enzyme complex that hydrolyzes ubiquitinated proteins, and the subunit of the proteasome, Rpt2, has undergone
N-myristoylation modification in many eukaryotes, including budding yeast. Recent studies have shown that N-myristoylation of Rpt2
dynamically alters the localization of the proteasome in the nucleus and cytoplasm, which intricately affects UPS in intracellular localization.
In order to investigate the effect of N-myristoylation of Rpt2 on UPS, strains with mutations in the N-myristoylation site (Rpt2-G2A, Rpt2-G2Δ)
and without mutations (Rpt2-WT) in Rpt2 of budding yeast proteasome were selected. A comprehensive ubiquitin proteomics analysis bymass spectrometry revealed that the Hsp70 family, Ssa1 and Sse1, involved in the transport of cytoplasmic misfolded proteins into thenucleus, showed a significant increase in ubiquitination in mutant strains. Therefore, the ubiquitination of Ssa1 in the wild strain and the twomutant strains was evaluated by IP-WB using Anti-Ssa1 antibody and Anti-ubiquitin antibody.

Result0.25 ug of Anti-Ssa1 antibody was reacted with budding yeast lysate, and 25 ul of protein G beads from each company were added torecover the antigen-antibody complex, and Western blotting was performed after SDS-PAGE.
From the results of Western blotting, it was confirmed that ubiquitination of Ssa1 was increased in both mutant strains as compared with thewild strain, similar to the result of mass spectrometry. In addition, when the amount of Ssa1 contained in lysate was examined by mass
spectrometry and Western blotting in three yeast strains, it was confirmed that there was no significant difference among the three yeaststrains.
From this, it was confirmed that inhibition of N-myristoylation of Rpt2 has the effect of increasing ubiquitination of Ssa1.
In addition, the following points can be confirmed for FG beads and beads of other companies.
☆Ssa1 and ubiquitinated Ssa1 could not be recovered with the beads of other companies, but they could be recovered with a highrecovery amount with the FG beads.
☆Other companies have the same configuration of the (+) and (-) fractions, but the FG beads have a high S/N ratio of (+) and (-).

FG beads

Beads:

IP

Sample:123Anti-Ssa1:+++Competitor
1-

1

2

3+++1-

Sample1: Wild type
2: Mutant (Rpt2-G2A)
3: Mutant (Rpt2-G2Δ)

(kDa)
250
Ubiquitinated Ssa115010075Ssa150
A. Kimura et al.
journal of proteomics 1 (130):33-41 (2016)
doi:10.1016/j.jprot.2015.08.021

37

Western blotting : Anti-ubiquitin

TAMAGAWA SEIKI CO., LTD.

http://www.FGbeads.com

Materials
aterials and method
Mand method■Materials
1. Protein G beads
2. S.cerevisiae lysate
3. Lysis buffer (20mM HEPES(pH8.0) , 9M Urea)
4. Protease inhibitor cocktail (from Nacalai Tesque)
5. PR-619 (Life Technologies)
6. Triton buffer (1% Triton-X100, 20mM Tris-HCl(pH7.4), 150mM NaCl,2mM EDTA)
7. Anti-Ssa1 antibody (from Santa Cruz)
8. SDS sample buffer
9. Anti-mono- and polyubiquitinylated conjugates antibody (FK2H) HRPconjugate (from Enzo Life Sciences)
10. PBS-T buffer (137mM NaCl, 2.7mM KCl, 8.1mM Na2HPO4・12H2O,
1.5mM KH2PO4, 0.1% Tween 20)
11. Blocking buffer (5% skim milk in PBS-T buffer)■Method 1 (Lysate Preparation)
1. Yeast culture
Culture yeast for 3 days at 30oC in synthetic complete
medium lacking leucine (SC-Leu).
2. Harvest and homogenize
Centrifuge at 3000×g for 10min, wash twice in sterilewater, and resuspende in lysis buffer with proteaseinhibitor cocktail and 50uM PR-619.
Vortex with glass beads and centrifuge to obtain lysate.

■Method 2 (Immunoprecipitation)■FG beads® information
Product nameProtein G beads

Product number

TAS8848N1173

Storage temperature

2-8oC

Storage buffer

10mM HEPES(pH7.9), 50mM KCl,
1mM EDTA, 10%glycerol

Size of beads

190nm ± 20 nm

IgG binding capacity>100ug mouse IgG /mg of beads■Methods 3 (Western blotting)
1. Perform SDS-PAGE.
2. Transfer the protein from the gel to a PVDF membrane using
Trans-Blot® Turbo TM.
3. Block the membrane with the Blocking buffer for 120min atroom temperature.
4. Dilute the Anti-ubiquitine antibody with the Blocking buffer to1/1000.
5. Incubate the membrane for 120min at room temperature.
6. Wash the membrane with PBS-T buffer 3 times.
7. Detect with a chemiluminescence substrate.

1. Lysate dilution
Add 9 volume of Triton buffer to lysate.
2. Reaction
Incubate lysate containing 125ug of total protein and 0.25ug
of Anti-Ssa1 antibody at room temperature for 60min on
microtube mixer.
3. Collect antigen-antibody complex
Add 25ul of Protein G beads, incubate at room temperaturefor 60min on microtube mixer.
4. Wash
Separate magnetically and remove supernatant.
Wash beads with Triton buffer 5 times.
Separate magnetically and remove supernatant.
5. Elution
Add SDS sample buffer and resuspend beads.
Incubate for 10min at 70oC and remove the beads.

TAMAGAWA
SEIKI
CO.,
LTD.
TAMAGAWA
SEIKI
CO.,
LTD.

http://www.FGbeads.com
http://www.FGbeads.com