Bitan:brain protein extraction

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Tanya Liu 4/4/11, updated Aida Attar 8-2-12

Brain extractions

1) Make solutions (TBS, TBST, 5 M GuaHCl) and add 2 COMplete protein inhibitor tablets (Roche) + 1 phosphatase inhibitor tablet (Roche) into 25-50mL of TBS and TBST

2) Take out brain parts & put in order -remember to keep on dry ice at all times or else will have condensation

3) Weigh empty microcentrifuge tubes, put brain parts in, weigh tube again

4) Note down brain weight and multiply by 5-7 to get total volume of buffer (ex: TBS) to add to brain

5) Break apart the brain with mortar & pestle followed by tip sonication a. Remember sonicate on ice and do short bursts (less than 3 secs)

6) Let sit on ice for 15min

7) Put solution in ultracentrifuge tubes a. Tubes need to be very balanced.

8) Put tubes in ultracentrifuge a. Spin at 60K (157,000g) for 15 min b. Press Door to open, depress center latch, close door, press Enter for program, press Start. Wait for temperature to go down. Wait until spinning at 5k before leaving the centrifuge c. Wipe down inside of tube holder with kimwipe

9) Take out supernatant and put into labeled tubes (ID#+L for lysis fraction) a. Make sure all tubes for all 3 fractions are siliconized tubes

10) Add in volume of next buffer (ex: TBST)

11) Sonicate pellet, let sit for 15min

12) Put into ultracentrifuge

13) take out supernatant and put into labeled tubes (ID#+T for triton fraction)

14) add in volume of next buffer (ex: 5 M Gua HCl)

15) sonicate pellet

16) put into labeled tubes (ID#+G for guanidine fraction)

17) put Guanadine fraction into rotator machine overnight at room temp,

18) put Lysis and Triton fraction in -80 freezer,

19) next day, put G fraction in -80 freezer (will need to desalt before running gel: Zeba *Bitan:SpinColumns from thermo scientific)

20) Solutions:

Stock 100mM trisHCl→MW is 157.6g, so make 0.788g in 50mL H2O

Stock 300mM NaCl→MW is 58.44g, so make 0.8766g in 50mL H2O

working TBS→10mL of 100mM trisHCl + 25 mL of 300 mM NaCl + 15 mL H2O→pH 7.4

TBST→1% triton x100

5M guan HCl→MW is 95.33g, so add 23.884 into 50mL of TBS→pH 8

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