First time I post something in the methods mail list (very exciting)
I am working with a CIB1 (calcium binding protein involved in cell
development, migration and differentiation) ortholog(F30A10.1) in C.
elegans. I was looking for any quick and easy calcium binding assays that I
could use as a possible activity assay.
Also question number 2:
would anyone know of a reason why WB would not work using nitrocellulose as
blotting membrane, as opposed to PVDF? Blots of this protein work with PVDF
membrane but do not work with nitrocellulose. Any ideas why?
In article <[Only registered users see links. ].net >, "Jose Olucha" <[Only registered users see links. ]> wrote:
Depends on A LOT of things! The easiest is to do native gel
plus/minus Ca2+ (i.e., gel shift). The traditional is radioactivity-based
- either equilibrium dialysis or gel-filtration. If the binding is very
strong, filter binding assay will work fine. For some freaky cases,
even blot overlay with Ca45 will do. If the protein is small and you
have a lot of it, NMR should detect Ca binding qualitatively.
The high tech is to do microcalorimetry - which, when executed
carefully, has a potential to be very quantitative. .
Perhaps your protein is too hydrophilic. Increase methanol % when