no bands in western blotting!!!!!!!!!!!

hi guys,
i am working with a protein which is 10kda.i am not able see a band at the expected region in westernblotting-things which i hav used
1.amersham ECL NCM -poresize 2u
2.0.1%SDS in blot buffer
3..63A for 30-45 min
4.0.3% Tween-20 in PBS for wasing and blocking
plz suggest me few things abt were i am going wrong.


[Only registered users see links. ]

In article <[Only registered users see links. ]>, [Only registered users see links. ] wrote:

As you don't mention controls, it is impossible to eliminate
any of the zillion possible reasons. However, my first reaction
is that the culprit is 0.1% SDS. 10 kDa is small enough to run
through membrane. On the other hand, at least something
should have remain bound, so you are also might have
problem with sensitivity.

DK

<[Only registered users see links. ].uk> wrote in message
news:[Only registered users see links. ]...
expected region in westernblotting-things which i hav used
[Only registered users see links. ]

First thing I would try is loading more protein on the gel. Then, higher
concentration of the antibody. Do you see other proteins being transferred
to the membrane? You can quickly stain/destain the membrane using SDS-PAGE
Coomassie staining solution. If you don't see protein bands, that means the
transfer did not happen (wrong membrane?). Usually, TBS or PBS with 0.1%
Tween-20 works fine. 5-50 ng of protein per lane, 1/10000-1/500 dilution of
0.2 mg/ml antibody are the usual working ranges. One more IMPORTANT thing in
case nothing works: your antibody may recognize the protein in native,
folded state and would not recognize the denatured, linear protein.
Solution: try another antibody (e.g., one raized against terminal peptide
sequences of the protein). As a last resort, in case another antibody is not
available, you may try renaturing the protein right on the membrane (depends
on whether your protein is a monomer in nature). There must be methods for
doing this somewhere out there, although I never tried it myself. Generally,
you would do the same thing as for in-solution renaturation.
-Emir