Dot Blot

Materials
	Nitrocellulose membrane / Whatman paper
	TBS-T
	Ponceau S
	Blocking Solution
	First and Second Antibody
	ECL solution and H2O2

Procedure

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• Whatman paper and membrane have to be wet  first whatman on dot blot, followed by membrane. Top of dot blot holder has to be firmly secured with screws on the lower part!
• Apply 50 µl of probe with equivalent amount of protein in dots
• Start vacuum till solution is through membrane
• Dry membrane for 10 min
• Stain membrane with Ponceau S for 1 min, short destain with TBS-T to check, if same amount of protein is on the blot, make a copy or scan blot
• Complete destaining of membrane with TBS-T
• Block the membrane for 2h with blotto at room temperature or over night at 4°C
• 1. antibody, over night at 4°C or 2h at room temperature
• Wash 3x for 10 min at room temperature with TBS-T
• 2. antibody, 1h at room temperature
• Wash 3x for 10 min at room temperature with TBS-T
• 10 ml ECL solution + 3 µl H₂O₂ for 2min at room temperature
• Develop membrane