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urgent-W.B visualisation on kodak film - (Jul/09/2011 )

Hi,
I did western blot on new antibody not yet tested, I developed the with Ecl plus using kodak film, I expose the film for 5 min, when I use the machine to develop my film, it was completly dark, I did another exposition of 10 min of another film, the film is black as the previous, I decided to do another one, after 15min, I saw my protein of interest at the right molecular weight, but the bands are white and the film is black huged background. the opposite of what I should see, black band on white film.
The film is new one and the filter is PVDF, I checked the signal on the filter, there is a signal all over the filter!!
anyway, I decided to develop the same western blot with The storm machine, I see signal of my protein, saturated becos all the bands have white intense color!!!
I supposed that the problem is the concentration of the antibody, either primary, I use higher concentration of the primary antibody noted in the datasheet. also I used 1/5000 for the HRP-Rb secondary antibody.
Iwasnt convinced about the signal, I decided to stop, and postpone this to another day, (2days later), I kept the filter at 4C. then, I decided to chek again since Im sure about the conditions of the western blot I did, everything was ok! a part from the primary antibody that I use the first time. I dont know if the background could be due to PVDF! I took the filter wash several times, and then use ecl plus again (5min)on the same filter, I did 20 sec.exposur with the film,I develop and see bands of my protein in each lane, with intense signal, but always the background all over the film, anyway its better than the first film I got 2 days ago. I decided to wash a bit the filter with PBS tween, dry a bit and put again in the cassette, exposure of 15sec, develop the film, the result was very good, clean and intense signal and the film was clear, no darkness at all.
My question is whether its possible to consider this result and I can qantify the proteins by doing actin on the same filter.
thank you for your prompt interaction

-luciana-

We had a simillar problem when someone accidentally substituted northern blot membrane for WB membrane in the box. Lab trolling at it's best.

-EvilTwin-

you appear to have non-specific binding of one of your antibodies to the blocking agent, cleared by washing with tween. you may want to include tween in all of your washes and antibody solutions (we do).

the blot should be valid after probing for actin.

-mdfenko-