User:Mar/Notebook/2007-7-23
From OpenWetWare
Jump to navigationJump to search
siRNA nucleofection (cont) [1]
re-blotting for actin
1:5000 GAM 1.5hr: much weaker MagicMark
second PAGE and blotting for ErbB4
Electrophoresis + Transfer
Concentrated extracts were loaded in 14µL:
#2 #3 MM+SB (1+5µL in 10µL) #4 #5
- Run and transferred onto Nitro (1hr)
Primary ErbB4
- Blocked 1hr in Casein and put o/n in 1:200 anti-ErbB4 (Abcam)
- washed 4x15' and put into 1:6000 DAR for 1hr, washed
- visualized on Fuji - ~strong bands at ~60kDa, none higher
Primary Actin
- Blocked 5' in Casein and put for 1hr in 1:60000 anti-pan-Actin (Neomarkers)
- washed 4x15' and put into 1:5000 GAM for 1hr, washed
- visualized on Fuji:
- actin is balanced between lanes!
- MagicMark too weak as compared to actin (in this dilution of the secondary)
Italic text