Blotcha

There are instances that make me wanna believe in God, not because miracles happen (if they do i.e.) but because some things are so inexplicable. They are so till the time I nail em down and hammer out the reason for the mystery and then I'm back to being an atheist.

So I couldn't get this blot right for almost a year now. I've done western-blots for the protein I work on many a time but that's not a big problem since antibodies have very high affinities for their targets; they recognize diminutive quantity of target that gets blotted. However N-terminal sequencing (NTS) was another story as it required a reasonably high amount of protein to be blotted on to the PVDF membrane. By fluke, I got it done once but that a very high quantity of pure protein and somehow managed to travel from the SDS-PAGE to the PVDF membrane.

Today morning, I gave up. The small quantities of proteolysed fragments just wouldn't blot despite my tenacious effort to do so for the past one month. After much deliberation, decided to look for some other way to get the NTS of the fragments and expressed my frustration to my guide. He suggested I check the literature on an alternative method. As I sat on the computer I thought I'd check out electroblot-troubleshooting. In the sea of results, I found a pdf that I sensed would be of some use and as I was perusing through it, came across a line and read no further. I sprinted across to cast the SDS-PAGE gel and get the transfer buffer ready. It consumed my whole day and I was mostly quiet, working but never doubting for a second that success was just a few hours away.

Six hours down the line, lo and behold, the most beautifully protein-blotted PVDF membrane that our lab has ever produced. It made my guide spring up from his seat. It made one of my labmates visibly upset (not everyone will be happy at your success), and others happy but happy or sad, they all came to me to inquire about the protocol. After having shared my knowledge, I called it a day.

A big hurdle crossed today. Now I'm looking forward to designing mutants and hopefully get one that works wonders and some poor man's life can be saved. Makes me feel so good about working hard on this project.