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Bands disappear after longer exposure - (Aug/09/2011 )

Hi everyone, I've had some interesting things happen with a Western recently and was wondering if you guys have any idea what went on.

I ran a Western with several stable clones I had selected and probed for gapdh (had had trouble detecting protein of interest but that's a different story). After a 2 minute exposure two of my clones showed strong staining (these were on the RHS of the gel), I then exposed the same membrane for 15 minutes and these two bands disappeared and the rest of my clones appeared! Nothing was changed during this time, all I did was remove the original film and place the second film on top. After this I washed the membrane, re applied the chemiluminescent reagents and repeated the exposures and had the same thing occur, these two bands appear and then disappear.

Details about the Western:
~15ug DNA loaded
Ran for ~2 hours at 120V
Transfered O/N at 40V at 4 degrees.
Blocked in 10% milk in PBST for 1 hour (shaking)
Primary and secondary ABs incubated for 1.5 hours with 3x 5 mins PBST washes in between, both concentrations 1:2000
Applied chemiluminscent reagents and developed as described above.

At the moment I'm just planning on running a fresh Western and seeing if I can get more normal results..
Thanks :)

--Fran--

-Fran- on Wed Aug 10 05:37:04 2011 said:


Hi everyone, I've had some interesting things happen with a Western recently and was wondering if you guys have any idea what went on.

I ran a Western with several stable clones I had selected and probed for gapdh (had had trouble detecting protein of interest but that's a different story). After a 2 minute exposure two of my clones showed strong staining (these were on the RHS of the gel), I then exposed the same membrane for 15 minutes and these two bands disappeared and the rest of my clones appeared! Nothing was changed during this time, all I did was remove the original film and place the second film on top. After this I washed the membrane, re applied the chemiluminescent reagents and repeated the exposures and had the same thing occur, these two bands appear and then disappear.

Details about the Western:
~15ug DNA loaded
Ran for ~2 hours at 120V
Transfered O/N at 40V at 4 degrees.
Blocked in 10% milk in PBST for 1 hour (shaking)
Primary and secondary ABs incubated for 1.5 hours with 3x 5 mins PBST washes in between, both concentrations 1:2000
Applied chemiluminscent reagents and developed as described above.

At the moment I'm just planning on running a fresh Western and seeing if I can get more normal results..
Thanks :)

Hola, it seems that your developping agent is poorly stable, I would try use one from other lab or add 2ul of H2O2 to 5ml of mine. Wait for more opinions . Buena suerte

-protolder-

Sounds like a flash - too much activation of the bright bands you are seeing initially, causes the HRP (presuming it is HRP) to "burn out" somehow. At least I seem to recall hearing about something like this happening before.

-bob1-

Thanks for the responses, it could be the burning out but I'm not sure.. Also it's strange that I did get different intesnity bands as I loaded equal amounts of protein (Bradford) and I was looking at beta tubulin!

--Fran--

Hmmm, check the reference range for the bradford assay - it only works for quite a low and narrow range of concentrations.

-bob1-