Immunoprecipitation + Western blot

(for co-immunoprecipitation or concentrating the sample before Western)

see Strahl-Bolsinger et al (1997) Genes Dev 11:83 SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast.

1. Start a 5 ml culture of the strain(s) and grow 6-12 hours.

2. Measure OD600. Determine the amount to add to 50 mls so the OD600 = 1 when you want to harvest cells the next day. (Note: if you are not comparing protein levels between different strains, you can make 15 ml cultures, skip the protein concentration measurements and precipitate everything in step 10)

• Use N_t / 2^g = No

• N_t is the OD600 you want at the end, and g is the number of generations the cells will go through between starting and harvesting the culture and No is the OD600 at which to start the culture.

• 1.3 hour = generation time for W303 non-mutant in YP5%D at 30C

• When you have determined the OD600 at which to start, determine how much to dilute the starting culture to get 50 mls at that OD600.

• Shake overnight

• If precipitating Cat8 or Adr1, derepress in 0.05% glucose for 6 hours before harvesting

3. Harvest when you have 50 mls at OD600 = 0.6-1.5 (approximately 2x10⁷ cells), by spinning 2-3K, 5'

4. Discard sup, wash pellet in 25 mls cold TBS

• Keep everything cold from this point on

• Pellets can be frozen at -70

5. Resuspend pellet in 400µl lysis buffer with protease inhibitors and PMSF added right before use. Transfer to an Eppendorf tube

6. Add an equal volume of glass beads and lyse.

• Vortex for 3 x 2 minute sessions on the cold room vortex, with 2 minutes on ice in between OR bead beat 2 x 30-45 seconds, setting 4 m/s

• If using the bead beater, keep the volume in the tubes under ~600µl

7. Spin 10K, 5' 4C, transfer sup to a new tube with PMSF

8. Spin 10K, 15' 4C, transfer sup to a new tube with PMSF

9. Measure protein concentration with Bio-Rad assay

• This doesn't need to be on ice, but the samples should be

• Dilute Bio-Rad mix 5-fold with water

• Set up 1 plastic cuvette for each sample plus 5 for the standards

• The standards are 0, 2, 5, 7 and 10 µl of 1 mg/ml BSA

• Make a small 10-fold dilution of each sample and measure 2µl

• Add 1 ml diluted Bio-Rad assay to each sample and standard cuvette. Read at OD595, zeroing the spec using the 0 standard.

• use a protein assay Excel sheet to determine protein concentrations

10. Immunoprecipitate

• Strahl-Bolsinger uses 400µl extract + antibody (Ab) + 250 units DNaseI for 3hours 4C

• For Adr1 or Cat8 use 1 mg lysate + 2 µg polyclonal anti-HA or 6 µg monoclonal anti-myc (optional: + DNaseI for 3 hours, 4C)

11. Add 60µl protein A sepharose (PAS), 1h 4C on a shaker or rocker

• Wash the PAS before use. Aliquot using a blue tip and shaking frequently.

12. Wash the PAS pellet 3 x 5 minutes with 1.4 ml lysis buffer.

• Spin a few seconds, remove sup, add lysis buffer and leave on ice, shaking occasionally.

13. Add 60µl Invitrogen protein sample buffer with ß-mercaptoethanol, heat 70C 10'

14. Spin 10' full speed, room temp, then transfer sup to a new tube

• Samples can be frozen at -70C but re-heat and spin before loading.

15. Run on a gel and do a western.

Lysis buffer: 50mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40.

PMSF 174.2 mg/ml in DMSO = 1M (1000-5000X, add before use, WEAR GLOVES)

protein A-sepharose CL-4B beads (Pharmacia 17-0780-01)

swell overnight 4^oC in TBS + 0.05% sodium azide

wash 3 times TBS + 0.05% sodium azide

resuspend ~1:1 bed volume: volume in TBS + 0.05% sodium azide, store 4^oC, wash before use