How is the conformation of proteins in a nitrocellulose membrane in a Western blot?

if you did an SDS gel when you separated it by size on your acrylamide gel than it should be denatured. typically you boil your protein in SDS as well right before loading. this denatures the protein very well (I usually load between 10-50 ug of protein). So your antibody probably isn't the issue. one little control I like to do after my transfer prior to blocking is to wash my gel in PonceauS (from Sigma) which will quickly and reversible stain for all the protein. It washes out in your subsequent TBS/PBS rinses and will tell you how much total protein is on your nitrocellulose (or PVDF) membrane. It also is really good to see if you had an air bubble where you expect your band. Did your ladder come through on the transfer just fine? do you see any non-specific bands on your blot if you over develop/expose? these things help trouble shoot a western.