Hi there.

I'm having "fade out" problems with my westerns.
I currently incubate in plastic pockets and have noticed areas on my membranes which gradually fade - almost like my antibody solution isn't getting to my membrane. Has anyone else seen this?
I've tried altering most other things - antibody dil., incubation time, etc...
I know that tube incubation can be done. Does anyone know if thats a better method?
Thanks.

What do you mena by fade?

Rb

I put the sealed plastic bag between 2 glass plates.

doing this you avoid this fading problems in the corner of your bag!

greetings

jenny

I too encase the membranes within a plastic pocket, I then will incubate them by hand for 60 minutes. Apparently a lot of labs use this technique, I think in part the warmth of your hands helps to incubate the antibody. A sort of gentle massaging motion will suffice, while ensuring to flip and rotate the membrane every so often.

If it is a mini gel size then I use a 50mL Falcon tube on a tube roller / tipper. If I'm using an antibody that is inexpensive or home-made (hybridoma SN) I use a Philidelphia cream cheese tub that is almost the same size as the mini gel membrane. Even with this you can use small volumes (about 5mL) and then use a rocker table.