ESO wikiadmin: Created page with "{| class="wikitable" |+ !Materials ! |- | |Nitrocellulose membrane / Whatman paper |- | |TBS-T |- | |Ponceau S |- | |Blocking Solution |- | |First and Second Antibody |- | |ECL solution and H₂O₂ |} === Procedure === ---- * 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 prot..."

2025-02-27T13:44:30Z

Created page with "{| class="wikitable" |+ !Materials ! |- | |Nitrocellulose membrane / Whatman paper |- | |TBS-T |- | |Ponceau S |- | |Blocking Solution |- | |First and Second Antibody |- | |ECL solution and H2O2 |} === Procedure === ---- * 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 prot..."

New page

{| class="wikitable"
|+
!Materials
!
|-
|
|Nitrocellulose membrane / Whatman paper
|-
|
|TBS-T
|-
|
|Ponceau S
|-
|
|Blocking Solution
|-
|
|First and Second Antibody
|-
|
|ECL solution and H2O2
|}

=== Procedure ===
----

* 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

Dot Blot - Revision history