In article (Dans l'article) <5dst5c$h53 at mserv1.dl.ac.uk>,
Stephane.Pasteau at pasteur-lille.fr (Stephane Pasteau) wrote (écrivait) :
> Hi everybody,
> I'd like to make immunoblotting on colony lifts to detect a protein
> expressed at the membrane of a bacteria. Have somebody a protocol to do
Here is our protocol:
Pipette 1 microL of your control on one side (back) of your membrane.
Put your membrane (we use amphoteric Nylon, Porablot NYAmp from Macherey Nagel)
on your Petri dish and let 1 hour at 37 degrees. (membranes can be
autoclaved if you want to go back to your positive clones).
Put your membrane 1 hour in PBS-milk (5% low-fat dry milk) at 37°C.
Wash 3 times with PBS-Tween 20 (0.05%).
Incubate with your antibody (conditions depending on your antibody;
usually 1 hour at room temp. is OK)
Wash 5 times with PBS-Tween 20 (0.05%).
If your first Ab is not HRP-labelled, incubate with the secondary Ab
(HRP-labelled) (1 hour at room temp. in the dark).
Wash 2 times with PBS-Tween 20 (0.05%) and once with PBS;
Do the revelation in the dark.
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