Western blotting - (Sep/08/2005 )
Hi,
I am doing westerns for a kinase which is about 60 KD. I have no problem at all with theprocedure but my only problem is that the band after developing are fuzzy and smeary. What I can do to get sharp and intact bands. I use PVDF membranes withmethanol and TBS. Thanks for u help.
-kuti-
Hi Kuti-
I do a lot of westerns and when mine are smeary I think it's usually because I got impatient and turned up the voltage to high on the gel run OR I let the transfer buffer get too hot during the transfer.
Not sure if this helps,
Good luck
Mountainman
-Mountainman-
QUOTE (kuti @ Sep 9 2005, 07:10 AM)
Hi,
I am doing westerns for a kinase which is about 60 KD. I have no problem at all with theprocedure but my only problem is that the band after developing are fuzzy and smeary. What I can do to get sharp and intact bands. I use PVDF membranes withmethanol and TBS. Thanks for u help.
I am doing westerns for a kinase which is about 60 KD. I have no problem at all with theprocedure but my only problem is that the band after developing are fuzzy and smeary. What I can do to get sharp and intact bands. I use PVDF membranes withmethanol and TBS. Thanks for u help.
Is it just your kinase that gives a fuzzy band? Or is it the whole gel? It would be helpfull if you could include a photograpch.
-Theo22-