Klymkowsky Lab On-line Methods
Photofishing & immunoprecipitation

Basta et al., in preparation.  



We use 5’ biotinylated  oligonucleotide was synthesized using a photocleavable phosphoramidite group, purchased from Phoenix Biotechnologies. 

The complementary oligonucleotide was unmodified and synthesized by Invitrogen.

After annealling (see DNA fishing protocol), DNA was incubated with streptavidin-agarose beads (Sigma) in coupling buffer for 1 hour at room temperature in the dark with constant mixing. The final concentration of DNA was 2.5 pmol/ml of beads.

Beads were washed twice with coupling buffer, and twice with 1x binding buffer (20mM Hepes, pH 7,9, 50mM KCl, 5mM MgCl2, 12% glycerol, 0.5 mM EDTA and 0.1% Triton X-100).

Embryo lysates are prepared by Freon extraction as described previously (Zhang et al., 2003).

600µl embryo lysate (30 embryo equivalents) in 1x binding buffer, 1mM DTT and 0.5mg/ml Herring sperm DNA was preincubated for 10 minutes at room temperature followed by 20 minutes incubation at room temperature in the dark with 100ml of DNA-streptavidin-agarose beads with constant gentle mixing.

Beads were recovered by centrifugation for 5 minutes at 3000 rpm, and washed twice with binding buffer.

DNA (and bound proteins) was released from beads by 3 minutes irradiation with Black Ray UVL-56 UV lamp (Ultraviolet Products Inc., San Gabriel, CA) at a distance of 7cm (emission peak 365 nm).

The suspension was spin-filtrated using Ultrafree MC filter unit, 0.22µm (Millipore, Bedford, MA), for 3 minutes at 5000 rpm

Fltrate (containing released DNA with bound proteins) was subjected to standard immunoprecipitation, typically 2 hours at 4°C in primary antibody followed by 25µl of protein A agarose (Sigma) overnight at 4°C.

After washing, beads were suspended in 2xSDS loading buffer and released proteins analyzed by SDS-PAGE and Immunoblot. 

Table of Methods  
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