User:Mar/Notebook/2007-7-23

= siRNA nucleofection (cont) =

re-blotting for actin
1:5000 GAM 1.5hr: much weaker MagicMark

Electrophoresis + Transfer
Concentrated extracts were loaded in 14µL: #2 #3 MM+SB (1+5µL in 10µL) #4 #5
 * 1) Run and transferred onto Nitro (1hr)

Primary ErbB4

 * 1) Blocked 1hr in Casein and put o/n in 1:200 anti-ErbB4 (Abcam)
 * 2) washed 4x15' and put into 1:6000 DAR for 1hr, washed
 * 3) visualized on Fuji - ~strong bands at ~60kDa, none higher

Primary Actin
Italic text
 * 1) Blocked 5' in Casein and put for 1hr in 1:60000 anti-pan-Actin (Neomarkers)
 * 2) washed 4x15' and put into 1:5000 GAM for 1hr, washed
 * 3) visualized on Fuji:
 * 4) *actin is balanced between lanes!
 * 5) *MagicMark too weak as compared to actin (in this dilution of the secondary)