This protocol describes the I-DIRT technique that helps to distinguish contaminants from the real interactors in immunopurified complexes. Here is the original paper describing the details of the technique.
PROTOCOL:
1. Prepare media
Media for Isotopically heavy Yeast- Reagents for 1L mix
Yeast nitrogen base without amino acids (Sigma Y0626)- 6.7 g
Synthetic drop-out media minus lysine (US Biological, D9515B) -2 g
mQ-H2O -900 ml
2. Autoclave
3. While autoclaving:
a. Dissolve lysine (Fisher BP386) into sterile mQ-H2O, sterile filter 80 mg in 1 ml
b. Dissolve d4-lysine (Cambridge Isotopes DLM2640) into sterile mQ-H2O, sterile filter 80 mg in 1 ml
4.Once the media has cooled down, add:
100 mg/ml ampicillin -1 ml
Lysine solution(either isotopically light or heavy)-1 ml
20% glucose (w/v) -100 ml
Grow tagged cells light and control untagged cells heavy. It is not necessary to use a lysine auxotroph in yeast as long as you stay in mid-log growth. Mix the final frozen cell pellets 1:1 and grind. Then follow your standard immunopurification protocol.
1. Put ~1-2g of pellets of frozen cells in the 50 ml stain less jars previously immersed in the liquid nitrogen until it stops boiling.
2. Close the jars. Do not screw the lid tightly.
3. Insert the jars into a Retsch Mixer Mill MM301, and beat for 5 times, 3’ at 30 beats per second. Between each beating immerse the jars in liquid nitrogen to cool. Efficiency of cell breakage can be observed in a microscope.
4. Transfer a cell powder back into 50 ml tube pre-cooled in liquid nitrogen. Be sure that there is no residual liquid nitrogen left in the tube.
NOTE> Cell powder can be maintained at –80ºC for long time at this stage.
For one reaction use
1. 500 ug of Antibody (Antiflag-M2 Monoclonal Antibody, Sigma F-3165, 5 mg of 4mg/ml solution). Antibody should be in phosphate buffer only! Glycerol and preservatives will interfere with coupling to epoxy groups activated on the beads.
2. 50 mg of Dynal beads (M270 Epoxy, 300 mg/vail)
NOTE> 50 mg of beads will be enough for 5-10 IP experiments. Add 5-10 mg of coupled beads to 10-20 ml of cell lysate made from several grams of broken cells. Do not store the beads for more than a month. Even though the beads will still work their quality will degrade over time.
1. Upon receiving antibody aliquot the antibody into 10 tubes. This should produce ~ 0.5mg/125ul of antibody solution. Note the exact volume of the solution.
2. Weigh 50 mg of dry powder of Dynal beads (~3×1e9 total # of beads) in a round bottom 2 ml tube.
NOTE> Do not use siliconized tubes!
3. Wash briefly with 2 ml of 0.1 M Phosphate buffer (pH ~7.5). Vortex 30 sec and put on the magnet. Remove the buffer.
4. Repeat washing with 2 ml of 0.1 M Phosphate buffer. Incubate for 10 min with rotation, place the tube on the magnet, discard the washing solution. The purpose of these two washing steps is to get rid of oxygen trapped on the beads.
5. Add ~125ul of antibody from one of the tubes (~500 ug/125 ul). Add the same amount of 2M ammonium sulfate. The final solution is 50 mg of beads in ~250 ul of 1 M of ammonium sulfate containing 500 ug of the antibody.
5. Seal the tube tightly and incubate @37 C with constant rotation. Couple for 24 hours.
Next day – washing the beads
1. Add ~2 ml of 0.1 M phosphate buffer, gently rotate the tube several time and put on magnet. Discard the solution.
2. Wash with 2 ml of 0.1 M Gly solution.
3. Wash briefly 5 times with 2 ml PBS +0.5% triton
4. Wash one more time with 2 ml of PBS +0.5% triton for 15 min with rotation. Discard the washing solution.
5. wash once with PBS without detergent.
6. Add PBS to one ml and store at 4C. For best results use all beads within one month.