Cell Lysis/Western/IP

Steven Finkbeiner, Departments of Neurology and Physiology, UCSF
http://gweb1.ucsf.edu/labs/finkbeiner/Protocol/protocol_list/Cell_Lysis.shtml
 
compiled by 6/95 by AJS from AB) Fresh 1X Lysis

Fresh 1X Lysis	Final	5ml	10ml
2X Lysis		2.5 ml	5ml
NaCl	0.25M	0.25ml of 5M	0.5 ml of 5M
Na3VO4	0.1mM	25l (ul) of 200 mM	5l (ul) of 200mM
NaF	50mM	0.5 ml of 0.5M	1ml of 0.5M
DTT	1mM	5l (ul) of 1mM	10l (ul) of 1mM
Aprotinin	3ug/ml	15l (ul) of 1 ug/ul	3l (ul) of 10 ug/ul
Pepstatin	2 ug/ml	10l (ul) of 1 ug/ul	20l (ul) of 1 ug/ul
Leupeptin	1 ug/ml	5l (ul) of 1 ug/ul	1l (ul) of 10 ug/ul
Okadaic		1:500
PMSF		35l	70l
H2O		1.75 ml	3.5ml

1X Lysis Buffer -- 4°C
Cool eppys -- 4°C put 4x # of tubes needed
PBS -- 4°C
Stimulation -- 37°C 10' NGF=100ng/ml à first aspirate 5 ml!
KCl=5 ml to 10 ml
Lyse -- on ICE TRAY Stop stimulation
asp. Media/wash 2XPBS/ +500l lysis buffer
scrape into eppy
incubate 45' 4°C à Vortex several times during
spin 10 secs 4°C
divide sample: ~400 l for IP/ ~ 100 l for Western
IP -- 1° Ab => 4°C 1 hour rocking
if 1° is not rab. à 2° Ab => 4°C 1 hour rocking (RaM 2l)
-- PrA/Seph 4°C 1 hour rocking 40l
-- Spin down 4°C 1'
-- Wash 3X: 2X Lysis Buffer (~500 l)/ 1X PBS

Sample Prep -- (a) IP: add (40 l) 2X Sample Buffer (1X V PrA/S)
-- (b) W: add (50 l) 3X Sample Buffer
Boil 5'

Run Gel -- 100 V through Stacking
100 – 200 V after (in Separating)

Prepare Transfer -- make fresh TB: 1L= 200ml 5X TBb
200 ml Methanol
600 ml H20
-- cut fresh nitrocellulose (11 X 17 cm) & Whatman soak in TB
-- 2 grids outside plate on Yellow Tape side
1 grid in middle – SMOOTH SIDE TO GEL!
Cushions
pour in TB until just wet
put 1 Whatman over Gel à smooth out
place onto cushion
Nitrocellulose over gel
2nd Whatman over Nitrocellulose à smooth out bubbles
4 cushions on top
2nd Grid – SMOOTH SIDE TO GEL!
Add more TB
2nd metal plate
place in tank: liquid should cover gel area Transfer use black battery charger: 24V 45'
After Transfer remove blot and place in container face up quick rinse TBST
Block  3% BSA/TBST or 5% Milk/TBST 1 hour RT shaking
1 °Ab dilute in 20ml: 3% BSA/0.05% NaN3/TBST
Wash pour 1 ° Ab back (RE-USE!) quick rinse, then 3X5' rinse TBST
° Ab HRPaM or HRPaR (depending on 1° Ab) dilute 1:20000 (1.25 l in 25 ml): 3% BSA/TBST 1 hour RT shaking
Wash quick rinse, then 3X5' rinse TBST
ECL 5 ml DuPont NEN White + 5 ml Dark bottle place blot into solutin for 1' + agitation put onto Saran Wrap and smooth out bubbles put fluorescent dot markers on for orienta tion