RE:no bands after developing
Posted by: user (IP Hidden, Unregistered user, )
Date: September 20, 2004 12:51PM
> > Hi,
I had the same problem with an antibody and milk. I could only get my band to show up if I increased the secondary to 1/1000 and used bsa either all the way through or blocked with milk then put my antibodies into 3% bsa in ttbs.
> > Hi
> >
> > I have used a stained gel[with Commassie] for western
> blot
> > wet transfer.I am confident that bands transferred on to
> the
> > membrane since I can visibly see them as the gel was
> > stained.
> >
> > However, after Ab & secondary Ab treatments with washings
> &
> > ECL kit ,I see absoultely nothing on my X-ray film.
> >
> > I have tried 15s,30s,1m,5m,15m exposures.
> > I used HRPconjugated Ab's.
> > Both primary & secondary Ab dilutions have been tried in
> 5%
> > non fat dried milk in TBS-T & also in 0.05% milk in
> > TBS-T.Blocking agent is 5% non fat dried milk.
> >
> > Any inputs on this one????
> >
> > Thank you
> > Sari
> >
>
> hi sari,
> i have had the same problem. maybe your AB are not specific
> enough. Also, see if your ECL kit is a little old.
>
 >  >
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