Subcellular fractionation (for 293 cells): C+N+HM Note: After harvesting cells, all steps must be performed on ice.

Amount of buffers used in this protocol are for cells harvested from a confluent 10cm dish. Harvest: 1. Aspirate medium from plates. 2. Wash 2X with ml 1X!"# and tr$psini%e with 1ml of 1X &r$psin '(&A. ). *ncubate at )+, for -. Neutrali%e with ./0ml of (1'123"#2!4#. 5. &ransfer cells to 1 ml conical tube. . #pin: 5,, 1000g, -. Aspirate supernatant. 6. 7esuspend pellet in 5ml ice/cold 1X!"#2 1m1 sodium vanadate 210m1 sodium fluoride. +. #pin: 5,, 1000g, -. Aspirate supernatant. .. 7esuspend pellet in 1ml ice/cold 1X!"#2 1m1 sodium vanadate 210m1 sodium fluoride. &ransfer all cells 81ml9 to microfuge tube. 0. &ransfer 1410th of the resuspended cells 8:100ul9 to microfuge tube. &his is the whole cell fraction for total protein anal$sis. 7emaining 000ul will be used for fractionation. 10. #pin all the above tubes: 5,, 1000g, -. Aspirate supernatant. Whole cell l$sate pellet is stored at /.0,. !ellet for fractionation is left on ice ;Do not freeze. Fractionation: 1. 7esuspend pellet in 1ml of h$potonic buffer 8"19. 2. *ncubate: 5,, )0-. ). <se 2+=142 needle and 1ml s$ringe to l$se cells. !ass the cells through the needle 10/1 times. Avoid formation of bubbles4foam. >old needle tip against the side of the microfuge tube. &his step re?uires some standardi%ation. 5. &o confirm l$sis, mi@ 5ul of l$sed cells with 1ul of &r$pan blue and observe on a glass slide under a microscope. Anl$ nuclei from l$sed cells taBe up the d$e, cells that are not l$sed remain unstained. 0 C of cells or greater must be l$sed for efficient fractionation. . #pin: 5,, 1000g, -. #ave sup and pellet. &ransfer :. 0ul of supernatant to fresh tube. Transferring the entire sup will cause contamination. #upernatant: ,$tosol 2 >14heav$ membrane 8'72 1itochondria9, !ellet: crude nucleus. 6. Nuclear fraction: 7esuspend the pellet 8nucleus9 in 1ml of nuclear isolation buffer 8"29. +. *ncubate: 5,, )- to -. (o not e@ceed incubation. .. #pin: 5,, 1000g, -. Aspirate supernatant. 0. 7esuspend the pellet in 1ml ice/cold 1X!"#2#D2#3 until pellet disintegrates. 10. #pin: 5,, 1000g, -. Aspirate supernatant. #ave pellet at /.0,. 11. Cytosol + HM fraction: #pin the supernatant: 5,, 10,000g, )0-. #ave sup and pellet. &ransfer :.00ul of supernatant to fresh tube. Transferring the entire sup will cause contamination.

#upernatant: ,$tosol !ellet: >1. 12. 7esuspend >1 fraction in 1ml ice/cold 1X!"#2#D2#3. 1). #pin: 5,, 10,000g, -. Aspirate supernatant and save pellet. 7epeat step 12 and 1) one more time. 15. #tore c$tosol and >1 fractions at /.0,. Processing fractions for regular !:

1. Nucleus: E$se with 7*!A, sonicate 2. >1: E$se with 7*!A, sonicate ). ,$tosol: Dacuum centrifuge with no heat until volume reduces to 1410th original volume 8:1. hrs9. 7otate at 5, till all precipitated salts dissolve. 5. W,E: 7*!A Processing sa"#les for $P an% !:

1. Nucleus: high salt buffer, rotate for 00- to 120-. 7econstitute salt concentration to isotonic levels before performing *!. 2. >1: 1C "riF4 1C N!50 ). ,$tosol: Dacuum centrifuge, 7otate at 5, till all precipitated salts dissolve. 7econstitute salt concentration to isotonic levels before performing *!.

H&P'('N$C )&S$S !*FF+, (!-)

(otal .ol/ Final Conc/ 10 m1 10 m1 ) m1 1 m1 1 m1 1 m1 1X 1 m1 012 ul +1+.. +. 1. 2.) +. 0.. 1. +. ).. -222 ul 0 + 10 2 ) 10 1 2 10

Stoc3 Conc/ 1 2 ) 5 6 + . 0 dd. >2A &ris 8!>+. 9 Na,l 1g,l2 Na)DA5 (&& '(&A &EA 8/20,9 !1#3 8/20,9 11 1 1 1 0.1 1 11 0. 1 100X 200 m1

N*C)+4, $S')4($'N !*FF+, (!2)

(otal .ol/ Final Conc/ 012 ul 6.0.) -222 ul 00+

Stoc3 Conc/ 1 dd. >2A

&ris 8!>+. 9 Na,l 1g,l2 Na)DA5 6 (&& + '(&A . &EA 8/20,9 0 !1#3 8/20,9 -2 NP52

2 ) 5

11 1 1 1 0.1 1 11 0. 1 100X 200 m1 10C

10 m1 10 m1 ) m1 1 m1 1 m1 1 m1 1X 1 m1 0. C

+. 1. 2.) +. 0.. 1. +. ).. )+.

10 2 ) 10 1 2 10 0

H$6H S4)( Nuclear )ysis !uffer for $P

3or nuclear protein e@tractionG 8from H. ". ,. Dol.2+0, No. 5 , November , pp. 5+265/ 5+2+1, 20059 for 1 ml 20 m1 &ris p> +.5 20 Il 11 &ris p> +.5 500 m1 Na,l .0 Il 1 Na,l 0. m1 '(&A 1 Il 0. 1 '(&A 0.2C N!50 20 Il 10C N!50 1 m1 (&& 1 Il 11 (&& 1 m1 Na)DA5 10 Il 11 Na)DA5 10 m1 Na3 20 Il 11 Na3 100X protease inhibitors 10 l 1X &EA 200 m1 !1#3 2. l 0. m1 !1#3 Add .) . Il dd>2A 4)) !*FF+,S M*S( !+ P,+P4,+7 F,+SH M4,8+,S F', P*,$(& 'F F,4C($'NS: ,$tosol: 'ps1 Nucleus: Eamin"1 >1: '7 ; ,alne@in 1itochondria ; D(A,1