User:Mar/Notebook/2007-7-23

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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
  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

  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:
    • actin is balanced between lanes!
    • MagicMark too weak as compared to actin (in this dilution of the secondary)

Italic text