Why are you blotting for so long? Usually wet transfer only takes an hour or so. I use semi dry and blot in 10 mins. If you have a very low voltage it will take a long time but this in itself may be a problem, leading to protein diffusion and fuzzy bands. Interrupting the current will only make this worse.

This is quite a difficult question...During transfer if there is a current interruption, usually proteins diffuse into the transfer buffer..the marker on the membrane is not sure sign of transfer..but you made the right thing in putting the transfer again...

anyway before putting the primary Ab on membrane, stain it with Ponceaux, maybe is everything ok..(if you are lucky!) 

 Was it a wet blotting or semi dry blotting you were performing? Normally it will take 1 to 1.30 hours for wet blot and for semi dry system 1 hours (there is also turbo blot from BioRad, 7 to 14 min is required). However, if you see the pre stained marker in your blot, that already give impression that transfer was done (though might not be that efficient!). You can check the gel by staining with commassie if  any protein still remaining there. Then simply go for down stream steps in your protocol and develop the membrane.  

Why are you blotting for so long? Usually wet transfer only takes an hour or so. I use semi dry and blot in 10 mins. If you have a very low voltage it will take a long time but this in itself may be a problem, leading to protein diffusion and fuzzy bands. Interrupting the current will only make this worse.

Thank you for the feedback guys! Staining with Ponceau showed protein :-)

(It was wet blot)

Once the protein has transferred to the membrane, it will stay there. Don't worry about left-over protein in the gel, there will always be some. Key is to never run anything overnight. You see now that power cuts can potentially ruin your experiment, a good reason to avoid overnight runs (no SDS-PAGE, not blot transfers, no antibody incubations). Keep all under control during the day. At best you can block the blot overnight and add the primary antibody in the morning for a 1h incubation.

As long as the transfer power pack was still ON, I think it should be fine. Usually, for large protein transfer, we did overnight transfer using 30 V.

Hi Elena,

As everybody pointed you, although transfer is highly dependant on the voltage and the type of blotting (wet or semi dry), the time of transfer you used should be enough for having good yield of protein on your membrane. I recommend you try shorter times in the future, in order to avoid some problems as difusion of the samples due to interruption of the voltage (I usually use 90-100 minutes, 200mA for wet transfer and it works perfectly).

But, anyway, if you have detected bands in Ponceau staining, it's a happy ending for your WB :)

Good luck in the future!

Jordi

Diffusion from the gel may have caused you to lose some protein when transfer was interrupted, but binding to the membrane should be irreversible. Transfer efficiency can vary, though, depending on sample concentration and amino acid composition. Probably a good idea to not use this blot for quantitative analysis, just in case.