View source for Dot Blot

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!Materials
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|Nitrocellulose membrane / Whatman paper
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|TBS-T
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|Ponceau S
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|Blocking Solution
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|First and Second Antibody
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|ECL solution and H<sub>2</sub>O<sub>2</sub>
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=== 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 H<sub>2</sub>O<sub>2</sub> for 2min at room temperature
* Develop membrane