IEF: Got stuck with my riddle - Problems with evaluation of my IEF blot (Jun/05/2008 )
Hi,
I've done an IEF to check phosphorylation of my protein of interest. There I found a dot with a shift of pI about 1.5 to the alkaline (same MW as my protein).
Has anyone an idea what it could be? I used ß-Mercaptoethanol in the loading buffer so I guess its not a konformational Isoform 
.
Please help (before I get bad dreams about mysterious dots on white nitrocellulose 
)
-jazim-
QUOTE (jazim @ Jun 5 2008, 07:19 AM)
Hi,
I've done an IEF to check phosphorylation of my protein of interest. There I found a dot with a shift of pI about 1.5 to the alkaline (same MW as my protein).
Has anyone an idea what it could be? I used ß-Mercaptoethanol in the loading buffer so I guess its not a konformational Isoform .
Please help (before I get bad dreams about mysterious dots on white nitrocellulose )
I've done an IEF to check phosphorylation of my protein of interest. There I found a dot with a shift of pI about 1.5 to the alkaline (same MW as my protein).
Has anyone an idea what it could be? I used ß-Mercaptoethanol in the loading buffer so I guess its not a konformational Isoform
. Please help (before I get bad dreams about mysterious dots on white nitrocellulose
)what is the reference to the shift of 1.5?
-The Bearer-
QUOTE (The Bearer @ Jun 6 2008, 11:25 AM)
what is the reference to the shift of 1.5?
Thats one of our problems: We're still working on a good reference. The problem is, that our antibodies against mammalian actin or Hsp70 didn't work with the yeast extracts I want to analyze. The calculated pI of my protein is 4.7 and the shift was estimated from a postdoc who did some IEFs but never before saw a shift like this. So it maybe also about 1 or 2. Is it possible at all, to get such a shift and if yes, what might it caused by?
-jazim-