Of cloned plasmids and stubborn proteins..........

My posts are getting fewer by the day,the reason? i am working my ass off in my lab.
This is specially for those computer geeks who think my work is not half as much hard work as theirs.
This is also for those who are still partying and holidaying.........the new year has long begun my friend and that too with a bang!! But for us meaner and humbler creatures of the dust its all about working on weekends..........:-(

Well for my six-month term project i am working with E.coli and Lambda phage.Its basically a study of certain protein-protein and protein-DNA interactions when an E.coli is infected by a phage.I had to initially construct plasmids which had my concerned proteins under study cloned tandem into them.

It was working as i had planned(after reading a dozen papers on the subject) and i was proud of my pace!!..........i always am at many other things as well!! Then then came my roadblock,after purifying the proteins i couldn't get them to express on the Western Blot.
Patience i thought would pull me through here but stubborn that these proteins are,i didn't get a single blot band on the membrane.To make things worse,i used a wrong reagent today and screwed it all up.

Now i am clueless as to what's my next step should be.............maybe i should prepare the primary and secondary antibodies for the blot again.
What the FUCK!!!!!!!!!! thats four days of my work,staying in that lab till 9pm gone.
Life's a BITCH!!!!