I'm having some trouble finding information about purifying rGFP using an Ni+2
agarose column. We just did a lab where we had to purify rGFP. I don't have a biochem textbook, and I wasn't able to listen to the lecture about this lab. I tried searching online for more information about it, like about the binding to the 6-his tag and about the protocol itself, but I'm not having luck finding anything. If you do know of a site which can help me, please post a link.. Because of not having a full understanding about this, I'm having trouble answering some questions in my lab report like:
- What is the underlying principle behind the lysing of bacteria that we used to isolate a crude extract of rGFP?
- Why did the pellet fluoresce green after you lysed the bacteria via a freeze-thaw cycle and spun out the debris?
- Two students performed similar methods of purifying rGFP as you did in lab today with the following exceptions: Jane Doe did all the purification steps in a large 4C room (called a cold-room) with breaking buffer [10mM Tris, ph 8.0; 200mM NaCl, 10mM nickel s ulfate]. John Doe did all his purification steps at room temperature with breaking buffer [10mM Tris, pH 8.0; 200mM NaCl; 500mM nickel sulfate]. Jane was able to isolate rGFP with the addition of elution buffer from the Ni+2 agarose column. John’s rGFP came off the column during the washing procedure. Why the discrepancy?
Any help would be greatly appreciated - thanks so much!