Embryo Lysates

• Take 25 embryos and place into 1.7ml centrifuge tube.
• Rinse once in lysis buffer (add ~ 1ml) and remove by aspiration
• Add 500 µL lysis buffer per embryo and homogenize with either P1000 tip.
• Add 1000 µL freon -- vortex
• spin in cold room, top speed - 10-15 minutes
• take the supernatant - either freeze at -80°C or use immediately for blot, immunoprecipitation, DNA-fishing or whatever else suits your fancy.
• For blots, heat at 80°C for 5 minutes (remember to add 2ME)
• Load onto gel. Normally 1 embryo equivalent will be more than enough per lane for whole lysates, although you will generally use more for IPs and DNA fishing.
  
  

  MK's modified lysis buffer

  50 mM Tris pH 8.0 150mM NaCl 0.5% NP40 0.5% Triton-X100 1mM EGTA 5mM NaF protease inhibitors

Immunoprecipitation with protein A agarose

• Use lysates prepared using freon extraction
• add 0.5-1 µL of antibody - incubate with end-over-end rotation for 1-4 hours at 4°C.
• add 40µL protein-A-agarose, incubate with end-over-end rotation for 1-2 hours at 4°C.
• recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm. Remove lysate by aspiration --it is ok to leave some liquid above the pellet.
• resuspend in your original lysis buffer (1.5ml) and wash for 5 min. at 4°C (with end-over-end rotation)
• recover beads by centrifugation, at 4°C, 2 min. at 2000 rpm
• resuspend in high salt wash buffer (buffer 2) and wash for 5 min. at 4°C
• recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm

High salt buffer:

• 50 mM Tris-HCl pH 7.5
• 500mM NaCl
• 0.1% Nonidet 40
• 0.05% sodium deoxycholate

Low salt buffer:

• 50 mM Tris-HCl pH 7.5
• 0.1% Nonidet 40
• 0.05% sodium deoxycholate

• resuspend in low salt wash buffer (buffer 2) and wash for 5 minutes at 4°C
• recover beads by centrifugation, at 4°C, 2 minutes at 2000 rpm
• remove supernatant as completely as possible without disturbing beads.
• spin again - remove supernatant
• add 1X SDS sample buffer ßME - heat at 80°C for 5 minutes - spin beads to bottom of tube
• load on gel!

Immunoprecipitation with protein A magnetic beads for silver stain/sequencing analysis

• prepare lysates (using freon extraction)
• absorb lysate by running it over a µMacs column (You can omit this step if you are not going to attempt silver staining or sequence analysis).
• add 0.5 µL of antibody - incubate with end-over-end rotation for 1-4 h at 4°C.
• add 50-100µL protein-A-magnetic beads (Miltenyi), incubate with end-over-end rotation for 1h to overnight at at 4°C.
• reequibrate fresh columns with lysis buffer (all subsequent steps are done at room temperature).
• run supernatant antibody beads over column.
• wash 4 x 200 µL with lysis buffer
• add 20 µL Hot 1X SDS-sample buffer - let sit for 5 minutes.
• add 50-80 µL hot 1x SDS-sample buffer and recover flow through. This is your sample! load sample on gel!

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