Cell Lysis/Western/IP

Cell Lysis/Western/IP

Steven Finkbeiner,Departments of Neurology and Physiology,UCSF

compiled by 6/95 by AJS from AB)Fresh 1X Lysis

1X Lysis Buffer -- 4℃

Cool eppys -- 4℃ put 4x # of tubes needed

PBS -- 4℃

Stimμlation -- 37℃ 10' NGF=100ng/ml à first aspirate 5 ml!

KCl=5 ml to 10 ml

Lyse -- on ICE TRAY Stop stimμlation

asp.Media/wash 2XPBS/ +500l lysis buffer

scrape into eppy

incubate 45' 4℃ à Vortex several times during

spin 10 secs 4℃

divide sample: ~400 l for IP/ ~ 100 l for Western

IP -- 1° Ab => 4℃ 1 hour rocking

if 1° is not rab.à 2° Ab => 4℃ 1 hour rocking (RaM 2l)

-- PrA/Seph 4℃ 1 hour rocking 40l

-- Spin down 4℃ 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 00 V after (in Separating)

Prepare Transfer -- make fresh TB: 1L= 200ml 5X TBb

200 ml Methanol

600 ml H20

-- cut fresh nitrocellμlose (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

Nitrocellμlose over gel

2nd Whatman over Nitrocellμlose à smooth out bubbles

4 cushions on top

2nd Grid SMOOTH SIDE TO GEL!

Add more TB

2nd metal plate

place in tank: liquid shoμld 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