Download 40 nm - PLOS

Proximity ligation assay (PLA) for signal enhancement and the detection of protein associations in situ
Different primary antibodies against Rad51p and Dmc1p or HA and mCherry tags and the corresponding secondary antibodies with attached DNA
oligonucleotides were used. Only if different primary antibodies attach to one and the same protein or two different proteins in close proximity,
complementing oligonucleotides A and B can ligate and be amplified to produce a detectable signal. For a principle of the PLA assay using
the Duolink II system see the manufacturer´s website at http://www.olink.com/ or Söderberg et al. (Nature Meth 3: 995-1000, 2006).
Mouse anti-Rad51/Dmc1
Anti-mouse + oligo A
Rabbit anti-mCherry
Rolling-circle amplification +
fluorescent nucleotides →
Microscopically detectable signal
Mouse anti-HA
Anti-rabbit + oligo B
Rabbit anti
anti-Rad51
Rad51
mCh
Dmc1
HA
Rad51
mCh
mCh
Dmc1
HA
HA
Rad51
Dmc1
Rad51
> 40 nm
HA
mCh
Dmc1
Dmc1
R d51
Rad51
R d51
Rad51
Dmc1p and Rad51p can be detected by a combination of antibodies
against the protein itself and the tag. This may work for single
molecules and for clusters of molecules. In this way,
y clusters of
Dmc1p were detected in the meiotic nucleus and clusters of
Rad51p in the somatic nucleus. Rad51p that is present in the
meiotic nucleus may be too disperse to elicit fluorescent signals.
In the same way, antibodies against mCherry and the HA tags
would produce a signal for adjacent Rad51p and Dmc1p molecules.
However, no such signal
g
((above the background
g
level in the cytoy
plasm) was detected, suggestingthat Dmc1p and Rad51p do not
cluster in the meiotic nucleus.