Western blot membrance blocking and storage - (Apr/24/2007 )
Hiya
Could somebody tell me whether or not you can store a blot once it's been blocked for more than 24 hours before adding the primary antibody, and if so, what conditions you do that in. My primary antobody is on order, but I have already run the gel and transfered to a membrane.
Thanks!
S
Could somebody tell me whether or not you can store a blot once it's been blocked for more than 24 hours before adding the primary antibody, and if so, what conditions you do that in. My primary antobody is on order, but I have already run the gel and transfered to a membrane.
Thanks!
S
yes you can, if your primary AB is made up in milk, then you just transfer blot to it but if you make the Ab in BSA then rinse your blot briefly maybe 5 mins in PBST and whack on AB, just make sure that your milk for blocking hasn't gone off though!
Thanks! One more question, how long can you leave your blot in blocking buffer before adding the primary antibody? My antibody will not arrive before the end of the week. Will it still be okay if I leave it until next week?
Could somebody tell me whether or not you can store a blot once it's been blocked for more than 24 hours before adding the primary antibody, and if so, what conditions you do that in. My primary antobody is on order, but I have already run the gel and transfered to a membrane.
Thanks!
S
yes you can, if your primary AB is made up in milk, then you just transfer blot to it but if you make the Ab in BSA then rinse your blot briefly maybe 5 mins in PBST and whack on AB, just make sure that your milk for blocking hasn't gone off though!
I've been storing blocked membranes for weekend without any problem, according to various sources blots can be stored for weeks (after blocking, eg. at +4 deg.C in PBS witn 0,005% sodium azide).
After blocking I just dry them on filter paper and keep at 4C until I want to use them. I use nitrocellulose, so I don't have to rehydrate like you do for PVDF. I don't even bother washing, even though I go from milk blocking to antibody in BSA solution, just throw it in.