In article <4sg4q3$mr6 at Trex.IenD.wau.nl>,
maarten.vanhelden at medew.ento.wau.nl (Maarten van Helden) wrote:
> hello,
>> I am looking for the proteins containing in saliva of aphids. I try to
> identify these proteins in gel electrophoresis, but untill now I always
have a
> strong smear during the migration, I have used several reductors (urea,
> mercapto, DTT, iodoacetamide) without any results, the smear is still
> appearing.
> Any informations about a procedure to remove this smear would be greatly
> apreciated,
> Responses can be emailed to the below address.
> Thanks,
I have no exp. with saliva or aphids, but here are some possibly useful tips:
- Gel overloading/excessive viscosity of sample: if you do not know the
protein concentration of your samples, try to estimate it (Bradford assay
for ex.) and then keep it around 1-5 ug total proteins loaded.
Alternatively, you can try serial dilutions of your samples to determine
the point (if any) where the smear disappears.
- pH problems: albeit the pH of the saliva should be in the buffering
range of your running buffer, you might want to check it once, if it is
too low, you can have troubles with the resolution.
- You can try also to precipitate the proteins in your sample by aceton or
TCA or EtOH and then resuspend them in a good buffer. This maybe will help
in getting rid of other distrubing components in the samples.
- Another very strong denaturant is GuanidineHCl. You could try it as well.
Hope this helps.
G.Maga.
