20µl of cell culture supernatant containing less than 10ng of TGFß gives a strong signal

• Wet Immobilon-P (Millipore) PVDF membrane in Methanol for 10 seconds

• Wash membrane with water for about 5 minutes, or until it sinks

• Equilibrate membrane in Transfer buffer for 5 minutes.
I use Tris-Glycine pH 8.3 with 15% methanol.

• Transfer at 10V/cm for 1 hour. I've also done 30V overnight then 10V/cm for 30 min.

• Stain for no more than 5 minutes in Coomassie Brilliant Blue stain

• Destain PVDF in 40 to 60% methanol with 5% acetic acid until background is white

• Photograph or scan or Xerox membrane

• Destain completely with 100% methanol and a bit of activated charcoal

• Block at room temperature in PBS-Tween (0.3% Tween-20) for 1 hour

• 1% BSA also works well to block

• 3% Non-fat Dry Milk Powder also works, but signal is somewhat weaker

• Use 3C7 at 100ng/ml diluted in blocking buffer or PBS-Tween. For the lot of 3C7 that I have, this is a 1:10,000 dilution.

• Incubate with agitation for:

1 hour at room temperature, or overnight at 4°C

• Use at 1 to 5µg/ml diluted in PBS-Tween. A 1:1000 dilution gives 1µg/ml.

• Incubate 1 with agitation for 1hour room temp or 4°C overnight

• 5 washes of 5 minutes with large volumes of PBS-Tween

• To detect 3C7 I use Biorad Goat anti Mouse IgG (H+L) Horseradish Peroxidase conjugated diluted 1:10,000 in PBS-Tween

• To detect R&D systems anti TGFß2 I use Sigma Rabbit anti Goat IgG (H+L) Horseradish Peroxidase conjugated, diluted 1:10,000 in PBS-Tween

• Incubate with agitation for:

37°C for 30 minutes, or overnight at 4°C

• 5 washes of 5 minutes with large volumes of PBS-Tween

• I use ECL kit (Amersham) or Renaissance kit (NEN)

• After final wash in PBS-Tween, drip dry membrane

• Immerse in detection solution for 60 seconds

• Place membrane between transparency sheets

• Expose to Kodak XAR film for various times, from 1 second to 10 minutes. As an initial try, I do a 60 second exposure.