Strange aspect of bands in Western Blot - (Dec/16/2008 )
Hi!
Sometimes when I reveal proteins through Western Blot procedure, I get as a result bands that are not well defined, and seems like small dots aligned together. I get it a lot when revealing phosphorylated Smad 2, but I have had it with some other antibodies. I don't know why it happens, and what causes it. Can anyone help me?
-cmcalvet-
QUOTE (cmcalvet @ Dec 16 2008, 12:15 PM)
Hi!
Sometimes when I reveal proteins through Western Blot procedure, I get as a result bands that are not well defined, and seems like small dots aligned together. I get it a lot when revealing phosphorylated Smad 2, but I have had it with some other antibodies. I don't know why it happens, and what causes it. Can anyone help me?
Sometimes when I reveal proteins through Western Blot procedure, I get as a result bands that are not well defined, and seems like small dots aligned together. I get it a lot when revealing phosphorylated Smad 2, but I have had it with some other antibodies. I don't know why it happens, and what causes it. Can anyone help me?
did you think in terms of unspecific binding of the antibody? if your Ab is polyclonal, cross reactivity is possible; try to improve your protocol for IB
-The Bearer-
QUOTE (The Bearer @ Dec 17 2008, 11:02 AM)
QUOTE (cmcalvet @ Dec 16 2008, 12:15 PM)
Hi!
Sometimes when I reveal proteins through Western Blot procedure, I get as a result bands that are not well defined, and seems like small dots aligned together. I get it a lot when revealing phosphorylated Smad 2, but I have had it with some other antibodies. I don't know why it happens, and what causes it. Can anyone help me?
Sometimes when I reveal proteins through Western Blot procedure, I get as a result bands that are not well defined, and seems like small dots aligned together. I get it a lot when revealing phosphorylated Smad 2, but I have had it with some other antibodies. I don't know why it happens, and what causes it. Can anyone help me?
did you think in terms of unspecific binding of the antibody? if your Ab is polyclonal, cross reactivity is possible; try to improve your protocol for IB
How can I improve it? Raising the concentration of blocking agent?
But it seems that is a problem with the running... I don't know...
-cmcalvet-