Western blot problems... - Western blot: Bad blotting? (Feb/28/2006 )

Hello

I am having some problem with the western blot I run. When I blot over from the gel to the PVDF membrane we use, I sometimes have bad transfer. The result is that once I incubate with my primary and secondary antibodies, my western picture looks like the signal has run all over the membrane. It is almost as if the proteins are binding badly to the membrane and are semi-washed away. Has anyone else experienced this problem?

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hi
first let me know have u keeping any +ve control if yes is it visualizing? if no in what dilution and what time u r keeping for incubation for primry and secondry ab and u r getting the bands atleast one time ?
what is your proteinnature is't hydrophilic or hydrophobic?
and u use nitro cellulose membrn also, in what way u r washing ? is it 1x tbst and u r pouring thewashing solution directly on membrane if yes dont pour directly it will wash every thing. and how much time u r keeping for blotting ? in what method and what volts if u keeping for toomuch time reduce the time of transfer and if ur sure that proteins are transfering by observe with prestained marker or ponceau reagent but not visualized by westrn then change the dilution of ab

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Hi
Yes, I have a positive control on my gel and I get a good signal but the signal seems to run so that I don't have 1 solid band. I dip the PVDF membrane in methanol and then equilibrate in water and transfer buffer. When doing the blot, I run at 100V for 1 hour. This problem doesn't happen every time, maybe every 3rd time. The problem doesn't seem to be only in my protein though as sometimes it is the standard that runs. I have attached a picture of a sample blot...

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Do you stain with Ponceau red after transfer?

how do you stain?
ECL?
are you sure there are no air bubbles between the membrane and the saran film when you expose the X-ray film?

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I use horseraddish peroxidase 2ndary antibody and then intensify the signal using a solution from Pierce (supersignal). After a 5 min incubation I expose the picture using a camera set on chemiluminescence for a couple of minutes.

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are you wetting your membrane properly prior to transfer?

i agree that it seems to be a problem with transfer, not with the rest of the procedure

have you tried new transfer buffers? have you confirmed the pH of every buffer you're using? are your electrodes in good working order? what transfer method do you use? have you tried an alternate transfer apparatus to ensure it's not merely mechanical?

I once saw very similar results with a semi-dry transfer, when the electrode had not been properly/evenly wetted prior to transfer step...

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Hi Jamie,

I think you need to eliminate some potential problematic steps in your next run. Sometimes its hard to tell whats going on without checking the process as you go.

I agree with the previous posts and think it has more to do the transfer and even perhaps the running step.

If you can;

Run two duplicate gels, but after running
- coomassie one gel (if this is ok it eliminates the running step),
- and transfer the other gel (making sure you soak the membrane prior to transfer and remove all bubbles between all layers of the sandwich...also making sure there is ample layers of whatman to create even pressure and no sliding of the gel).

After transfer coomassie the gel and ponceau the membrane. If these look fine continue with the ab detection.

Ohh and another thought.. when it happens every 3rd or so time are you using fresh reagents/re-used reagents..setting the gel on the day..ie is it fresh APS, reused running/transfer buffer. Perhaps if you jot down what you do exactly each time there might be a hidden cause that you do differently.

I hope you get it to work 100% soon.

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Are you wetting the PVDF with methanol before transfer?

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