915
edits
Changes
added links to buffers
==Materials==
*Transfer Buffer (200 mL Methanol, 100 mL 10X [[ Transfer Buffer ]] to final 1L volume)
*Transfer Apparatus, either Bio-Rad or Invitrogen
==Protocol==
#Run SDS-PAGE gel using [[ SDS-PAGE Running Buffer ]] and prepare diluted transfer buffer
#Make sandwich, ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
#Transfer 4h at 75V (in cold room) or overnight at 35V (on the bench).
#Carefully remove blot, stain with Ponceau solution and rinse with TTBS until all the red is washed off
#Block with 2% BSA in [[ TBST ]] or 5% skim milk powder in TBST for >1h
#Incubate with primary antibody (check for dilution) in 1 mg/mL BSA for >1h
#Wash blot every five minutes for 30 min with TBST.
#Drain excess buffer from blot and cover with ECL for about a minute
#Drain excess ECL from blot, cover with saran wrap and expose film
[[ Category: Western Blotting ]]
