so i have reached a dead end. - (Nov/21/2006 )
this is it. i dont know what to do.
Aim of my research: to construct a recombinant protein and cleave it in a test tube with a protease.
First approach: try E-coli. Cloning went fine but protein turns out to be non-soluble and degraded. So i try to get out only non-degraded part for cleavage using Urea gel (non-denaturing conditions) but no success.
Second approach: try mammalian. Using His tag vector from santa cruz, it seems to break when something is inserted into it (reported using different kind of samples by other students too).
Third approach: Try EGFP and just transfect into the cells to see if it's cleaved inside there. Cloning worked (confirmed RE digest and PCR) but no fluorescence. Not a frame problem i think.
Have no other vectors to clone into. The only simple choise i have is to use another EGFP and clone by single RE into the MCS. but i am afraid to start all over again. Something inside tells me to stop and think more. I have spend one year and had got NOTHING to publish. What would you advise me to do or concentrate on. any help HIGHLY appreciated, as i am soooooooo lost i have to submit this paper by next month! or there is noway i graduate..
This isn't my field of expertise but are there such thing as in vitro translation kits (i.e. kits for translating your protein in a cell-free system)? I believe this is possible, but whether there are kits for it I'm not sure.
kathy for which purpose u need to publish a paper within dec?? is it master's or doctor??
sorry i have no suggestions about ur problem,,,,,,try to discuss with your sensei a lot
I agree with T.Reesei, you need to have a long chat with your supervisor. I am not sure what kind of paper you need to write bu if its a thesis then I am sure you will be fine. You need to outline what you have done, why you think it hasn't worked and present potential solutions. Students are often rewarded for showing good scientific logic that involves following scientific process and thorough problem solving.
Is research $$$ an issue for you? Would GST-tag expression vector help in this case? GST-fusion protein tends to be more soluble and if you can get enough soluble protein, then you are back in the game. AP bio/Phamacia sells this vector. I believe some NEB, or Novagen vectors can also improve solubility of the recombinant proteins. Good luck to you.