Last night I shared my experience in protein purification with my roommate, who is specialized in studying protein. She laughed so hard that I think I should write down my story, which took place this February.
My PI asked me to purify a protein, a FRET sensor with HIS tag at the N teminus. He gave me a protocol out of his head, claiming this was what he had been doing for a decade and this is the protocol that worked for the previous graduate student, Abhi.
He asked me to start by preparing the bacteria soup in the volume of 100ml. Yet I can see no fluorescence in the tube after elution. He accused me of having bad hands so that I must have lost all the proteins during purification.
He then asked me to prepare the E.coli soup in 1 litre so that I could have more room to make mistake yet still yield purified protein. The bacteria soup and all these reducing agents smelled so terribly I had to wear a plague mask, but even that I still suffered from a terrible headache induced by the stench. Yet nothing worked.
He then accused me of not following the protocol religiously and asked me to have him watch me when I was doing my experiment.
I did it again. I showed him the bright yellow fluorescence (from the YFP in the FRET sensor) in the bacteria pallet yet nothing yield in my elution solution.
It was the day of recruitment. I remembered potential candidates walked in the lab, and I was on my plague mask. I had to apologize for the stench.
Later after dinner with candidates, he approached me and told me that he made a mistake. He mixed up the formula between elution buffer and wash buffer.
For the whole time, I had been, under his instruction and supervision, using elution buffer to wash my beads for purification.
I guess it is always a great story to tell.
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