EZ-ECL Kit, Cat.# 20-500-120, Protocol for Nucleic Acids

Action:

According to the usual protocols

Action:

Immerse the membrane in ddH2O and then in transfer buffer.
Transfer to the usual protocol

Remarks:

Use nitrocellulose or nylon membrane

Action:

Soak the membrane in 6xSSC
Dry for at least 30 minutes
Fixation by backing or by U.V. crosslinking
5 minutes at room temperature
room temperature
0.5-2 hours at 80ºC or 32 seconds 1200 erg.

Volume:

0.5ml/cm2

Action:

According to the manufacturer’s instructions
Use denatured biotin-labeled DNA probe

Volume:

0.1 ml/cm2

Time:

1-2.5 hours at 68ºC or 60ºC

Action:

Dilute the HRP-labeled secondary antibody (1:10,000-1:60,000) in TBS-T or PBS-T with 2% dried milk (w/v). incubate the membrane in the solution

Volume:

0.1ml/ cm2

Time:

1 hour at room temperature

Action:

Twice with 2xSSC, 0.1% SDS (Wash no.1)

Volume:

0.5ml/ cm2

Time:

2x15 minutes at room temperature

Action:

Immerse the membrane in Buffer A
Incubate the blot in 0.2% EZ-Block in Buffer A

Volume:

2ml/cm2

Time:

30 minutes at room temperature

Action:

Dilute the Streptavidin-HRP (1:2000-1:10,000) in 0.5% EZ-Block in Buffer A. Incubate the membrane in the solution

Volume:

0.125 ml/cm2

Time:

30 minutes at room temperature

Action:

Three times with 0.1% Tween 20 in Buffer A

Time:

3x10 minutes

Volume:

2ml/cm2

Action:

Mix equal volumes of EZ-ECL solution A&B

Time:

5 minutes

Volume:

0.1ml/ cm2

Action:

Incubate membrane in detection mix solution

Time:

1-3 minutes

Volume:

0.1ml/cm2

Action:

Remove excess detection mix, wrap in saran wrap and expose to film

Time:

0.5-6 minutes

Remarks:

Remove air pockets