western blot - missing expected band - (Mar/15/2006 )
I am new to western blot. Our aim is to prepare a sample for N-terminal sequencing by PVDF membrane. we digested the protein with trypsin to get a particular fragment (~35kD) and ran western plot and stained the PVDF by Ponceau S for 30 min and destained in water. The destained membrane clearly shows the standards bands as wells the other digested fragments (60KD, 25kD and 18KD) in correct places but did not show the expected one (45KD). There is no even light band. we tried with different stain with CBB, etc. The result is same.
we checked the SDS-PAGE gel, it is not prensent it, thus it should be moved from it. We wonder that where it is gone?
We ran two SDS page gels with identical conditions to check wether it is missing in the SDSPAGE gel itself. The SDSPAGE gel clearly shows all digested fragments including the 35KD.
How did we miss only that particular fragment (35KD) in western blot.
We don't know the sequence or nature (eg. charge) of that fragment.
We did not use any antibody or blocking agents because our aim is different.
could you please give us any suggestion?.
with advance thanks,
We identified the problem. we put 0.05%SDS in Buffere. It works fine.