PRODUCTION OF RECOMBINANT (BACULOVIRUS PRODUCED) IRS-1 PROTEIN

A) INFECTING Sf9 CELLS (STEPS 1-2 ARE STERILE)

**It is best to start an infection in the morning, since you will harvest 54 hours later (e.g 10 AM infection will be spun down to harvest at 4 PM and there are 2 hours of subsequent steps).

1. 10⁸ healthy (nice round shiny cells) Sf9 cells from spinner culture at 1-2 x 10⁶ cells per ml (count by hemacytometer: count one grid block consisting of 16 squares and multiply by 10⁴ to get cells per ml) are spun down in 50 ml conical tubes for 5 minutes at a setting of 3 on an IEC tabletop centrifuge (Kahn lab). Remove supernatant by aspirating.

2. Resuspend cells in 10 ml of HIGH TITER virus solution in one of the 50 ml conical tubes (count the infection start point here). Leave 1.5 hours in hood (capped), and then put it in a sterile spinner with an additional 90 ml TNM-FH media. Its a good idea to have seperate bottles of media for viral and non-viral work. Put spinner in Sf9 incubator.

B) HARVEST RECOMBINANT PROTEIN

3. After 54 hours of infection, pour infected cells (non-sterile from here on) into 50 ml conical tubes and spin down as above in IEC centrifuge. Aspirate supernatant, and resuspend Sf9 cells in 1 ml of ice cold lysis buffer:

Bac Buffer (1 M NaCl, 50 mM Tris 7.8) with: 50 ml

0.01 mg/ml Aprotinin 500 ul of 1 mg/ml stock in water

0.01 mg/ml Leupeptin 500 ul of 1 mg/ml stock in water

10 mM DTT 500 ul of 1 M stock in water

2 mM benzamidine 500 ul of 40 mg/ml in water (fresh)

2 mM PMSF 500 ul of fresh 35 mg/ml in EtOH

**EVERYTHING FROM HERE ON IS ON ICE OR IN THE COLD ROOM.

4. Transfer to 7 ml dounce with tight pestle and dounce 20 times (smoothly, no bubbles). Transfer to Ti 70 ultracentrifuge tube. Spin out debris for 1 hour at 55 Krpm at 4 C.

5. After the spin, there will be a pellet at the bottom of the tube and teh supernatant will appear layered. You want all of the supernatant, remove it into an eppendorf or 15 ml conical tube and mix by pipetting gently. Save 10 ul in 100 ul of LSB to make sure the infection was good.

6. Load the supernatant onto a large (SK 16/100- (Pharmacia) containing SK 300 HR media (Pharmacia) equilibrated in Bac Buffer). Run the column overnight at 4 ml/hour. Collect eluate in a 100 ml graduated cylinder. Begin collecting fractions (1-1.3 ml/fraction) the next morning after about 60 ml have gone through.

C) FRACTION ANALYSIS AND IRS-1 STORAGE:

7. After about 110 total mls have flowed through the column (50-60 fractions collected after initial 60 ml) you should proceed with fraction analysis: Take 20 ul of every third fraction (beginning with fraction number one. i.e. 1,4,7,10,13....) and place into an eppendorf tube with the appropriate fraction numbver marked on the tube. Add 50 ul of 2x LSB containing DTT to each tube and boil (hole in top of tube to prevent popping). Load the samples in their entirety onto two 7.5% minigels (1.5 mm spacers, 10 tooth combs) with prestained standard, also load 10 ul of the laemmli-ized supernatant from step 5. thus you will load 17 fractions: every third 1-49. If you feel crazy, since it is now early evening, you may run the minis fast (100 V stack, 200 V resolving). Otherwise running the pair overnight at 5 mA is good.

8. Transfer the gels to PVDF paper for 1.5 hr at 100 V (see notes in immunoblotting protocol).

9. After transfer, rinse the membranes briefly in deionized water and stain 2-3 minutes in 50% MeOH containing 0.1% Coomassie Brilliant Blue (R-250). Destain with 4-5 rapid (45 seconds) changes of 50% MeOH, 10% HOAc in water, shaking in your hand. Do NOT stain/destain in western blotting boxes, use the lid to a box of pipette tips.

10. Pool the 4 or so peak fractions (only two of which will appear on your gel). E.g. fractions 19-22. and distribute in 100 ul aliquots in eppendorf tubes marked with the prep date and other pertinent info (e.g. mutant?). Place aliquots immediately into -70 C. freezer.