So it turns 0ut that after all that time I spent doing dialysis, we may have come up with a better way to purify the HA-GMA. Rather than 10 days, this method takes one day. We tried to run a model reaction today using the new purification method, but after the second time purifying the HA-GMA, I added to much water and the product did not precipitate. So essentially, I wasted an entire days work, but oh well. No more dialysis!
Jul
06
This past Thursday I was trained on and used the Solid-Phase Protein Synthesizer (SPPS). It’s a neat machine. All you have to do is measure out the amount of each amino acid you want in your peptide into specialized vials, place them in the machine, tell it which amino acid to start with, input a few other options wrt the chemistry, and press run and the machine will build the peptide onto resin. The only problem is when your peptide is 48 amino acids long (as is our perlecan sequence) it takes awhile measure out the precise amount of each amino acid and activator. I think I spent a total of 8 hours Thursday and Friday measuring amino acids. Not fun. But it was worth it because the peptide had all weekend to synthesize (instead of wasting time during the week).
Jun
30
Finally finished up dialysis on the first HA-GMA samples. I then proceeded to filter the solution, and began freeze drying some of the samples, which will take another 3 days to complete. Furthermore I should be getting training on the Solid-Phase Protein Synthesizer (SPPS) on Thursday, which should be interesting.
Jun
24
Next time I run a dialysis of the HA-GMA, start with 5 membranes. It will make things easier and save time.
Jun
23
Light work day in the lab = more time to monitor the Woot off!! This time I will get the random bag of crap!!!!
Jun
22
The dialysis is taking longer than expected. It’s been 4 days already, which is the time it normally takes to purify the mixture, and there is still too much DMAP in the HA-GMA. As a result, we’re going try 3 more days of dialysis and see what that does. In other news, one of the membranes broke again. Fortunately we had three, so less of a loss was incurred this time. We split the solution into 4 membranes now. Hopefully that will be enough.
Jun
22
Here’s what I took away from the ethics seminar on Thursday: Ethical egoism is the ethical position that one ought act in their own self-interest, or as my RA used to say, “You have to make the decision that is right for you.”
Jun
18
The ethics seminar on Monday was quite interesting. We talked about some of the basic schools of thought in philosophy, how some people believe that the morality of a decision is based upon the good of the whole, while others believe in a moral code that dictates the morality of a decision. Given these two schools of thought, its easy to see why topics such as abortion, stem cell research, euthanasia, torture, capital punishment, etc., are such controversial issues.
In other news, after resynthesisizing the HA-GMA polymer, it has survived the first day of dialysis. I can see why the membrane lyzed the first time. We filled the membrane/bag with about half as much of the mixture as last time and after only one day of dialysis, the bag is already almost full.
Jun
15
In synthesizing the hyaluronic acid (HA)/glycidyl methacrylate (GMA) based hydrogel we’re working with it first takes 1 to 2 hours to dissolve the HA in water. Next you add dimethylaminopyridine (DMAP), tetra butyl ammonium bromide (TBAB), and GMA in that order and stir the reaction mixture under nitrogen atmosphere for 48 hours. Then we added 6 M HCl and let that stir for an hour, and then we added NaCl and precipitated the mixture in acetone. The reaction to this point took the better part of a week. Once we vaccuum filtered the precipitate, we placed it in a dialysis membrane set it in a 4000 mL beaker over the weekend to let the unwanted salts in the mixture diffuse from the membrane into the water. Unfortunately over the course of the weekend the membrane ruptured and the would-be hydrogel spilled into the water. We have two theories on why this might have happened. First of all, the bag might have been too close to the stir bar in the beaker, and repeated collisions might have caused the bag to rupture. Second the bag, being almost full, might have had too high a concentration of salts and so the bag rupture on account of the salts diffusing too quickly? Whatever the case, there goes a week’s work. Back to stage 1.
