Wednesday, 17 August 2011
Monday, 15 August 2011
End of the experiments, the write ups begin
Last week I finished all my lab work :D however this means I have 2 whole weeks to write up stuff and make presentations. So last week I tested using really small volumes (80ul) and it worked, sort of. Now all I need to do is suggest some improvements, no pressure then. I have soo much writing to do I'm glad I have these two weeks, and two presentations and one is infront of the people in the lab so they actually know what I'm talking about, gulp!
Friday, 5 August 2011
End of the protein prep :)
Hopefully today I will have purified some S1 from myosin. It has been a busy week making very large quantaties of buffer (25L is my record) and as of this morning when I assayed the solution I calculated the total mass of the protein to be a massive, an incredible 1.36 g. Apparently this is good, but I can't help but feel its a little small as I started of with aroung 750ml of myosin. But anyway get to fast freeze it in liquid nitrogen later, very cold liquids and me may be a dangerous mix but hey better give it a go. I am honestly happy that my protein no longer looks like a white, sticky, mush which being a sick minded individual made me think of... never mind. It is dawning on me that I only have 3 weeks left, which is a little scary. Still I'm sure I will have 3 more fun filled weeks of stoppe-flow :D
Sunday, 31 July 2011
All fine, all change
So my supervisor Mike got back on Friday from the US and said that all the flouroscein stuff I did was fine, YAY! However the ADP affinity went a bit crazy so may have to do that again at some point, bad times. But on a lighter note I was getting a bit tired of stopped-flow so I had a word and I get to do a large scale S1 protein purification this week, hurray. Its so large in fact that its going take all week, but should be fun, and I get to play with a really big chromatography column and make 15% gels and to runs etc. I'm really looking forward to doing it because its something different and it made me laugh that we are using large volumes for example we put 10l of water in the fridge on Friday and at one point we use a litre of Chymotrypsin we only get 3-4g of S1 out if we're lucky.
Jonathan
Jonathan
Tuesday, 26 July 2011
Testing the shot volume
So since my lasy post I have been varying the volume of the shot driven through the machine. I'm pushing Flouroscein against Flouroscein so that no reaction is observed but it will show a change in the florescence as the temperature changes. Before I was using a machine with a 60 ul cell, however this broke so I am now using one with a 5 ul cell. The advantage of this is that it is better suited for cold temperatures and I can use smaller shot volumes. The volumes I used where 20, 30, 40, 50, 60, 80, and 90 ul. However the traces of the florescence that I obtained only showed any temperature change at 80 and 90 ul, this would suggest that the device on the cell is cooling down or heating up the sample instantly from 20 - 60 ul. The way in which I can determine this is that if there is no temperature change then the trace will come out flat, if there is a heating up effect then the florescence will shoot up then return to the starting amplitude, if there is cooling then there will be a drop followed by a rise in amplitude. This was fairly dull as most of the time I was looking at traces of just horizontal lines, hopefully the rest of the week will be better as I am moving on to finding the ADP dissociation constant when mixed with S1, actin and ATP.
Thursday, 21 July 2011
My Placement so far
Hi, my name's Jonathan Walklate and I'm taking part in a summer placement at the University of Kent funded by the biochemical society. My aim is to test out modifications on High-tech scientific's stopped flow machines. I am currently in my third week and have only just gotten round to blogging, so I'm afraid this will be a long one.
week 1: So I started out by reading the literature around device and the technique's that I will need, and then I concluded my first day making my first ever buffer and having a demo on the stopped-flow machine. The first thing that I needed to get results on is to make sure that a modification works. In a normal stopped-flow machine the samples are held in syringes in a water bath so that the temperature can be controlled, they are then pump through tubes to a light cell where a florescent spectrometer can obtain the florescence of the mixture and therefore measure the reaction on a millisecond time scale. One problem with this method is that as the sample leaves the syringes it is no longer thermostatically controlled. The first modification that I was testing was a chamber between the water bath and the cell which is also thermostatically controlled. To do this I set the water bath at 10 C and the chamber at different temperatures from 5 - 45 C, I also used 4 different ATP concentrations to react with the skeletal rabbit S1 region of the myosin that binds to actin, (the actin was labled with pyrene which fluoresces at 365 nm) and then created 4 Arrhenius plots to show that the chamber worked. These results have been incorporated into a sales poster by High-tech scientific.
Week 2: I started of by testing temperature jumps using first pyrene actin and S1 against pyrene actin and S1 (PA-S1 ) going from 20 C to 20 C, this should have given a constant florescence but this didn't happen. So I tried Flouroscein, but again this didn't work. So stumped we moved on to carrying on the previous experiment by increasing the ATP concentrations used, I then plotted these results to find the second order rate constants and then also plotted these against 1000/temperature in Kelvin. I finished the week by completing this.
Week 3 to now: So I started this week by retrying the temperature jumps. This time still using Flouroscein we tested 3 different buffers, the one I had been using was a cacodylate buffer containing 100 mM KCl, the ones I tested where; 100 mM KCl Tris, 100 mM KCl KPi, and 100 mM KCl Imidazole. The best of these was the Imidazole buffer which gave the desired trace and also is affected by temperature compared to the others, giving more accurate results. So I managed to test two temperature jumps one from 20 C to 30 C and the other 20 C to 5 C. These both worked, but my aim was to obtain better results by having the same end point of 5 C, however today everything went wrong as firstly the values on the machine failed meaning the samples were not mixing in the cell so I obtained odd results. We then changed the machine for one that is better suited for low temperatures. So I started setting up my experiment this afternoon only to find another problem that the Mercury/xenon lamp had blown, so I had to waste half an hour for the lamp to warm up. Finally I managed to check this machine was working the same as the other, even though the cell was 20 ul in the first and the new one is 5 ul.
Jonathan
week 1: So I started out by reading the literature around device and the technique's that I will need, and then I concluded my first day making my first ever buffer and having a demo on the stopped-flow machine. The first thing that I needed to get results on is to make sure that a modification works. In a normal stopped-flow machine the samples are held in syringes in a water bath so that the temperature can be controlled, they are then pump through tubes to a light cell where a florescent spectrometer can obtain the florescence of the mixture and therefore measure the reaction on a millisecond time scale. One problem with this method is that as the sample leaves the syringes it is no longer thermostatically controlled. The first modification that I was testing was a chamber between the water bath and the cell which is also thermostatically controlled. To do this I set the water bath at 10 C and the chamber at different temperatures from 5 - 45 C, I also used 4 different ATP concentrations to react with the skeletal rabbit S1 region of the myosin that binds to actin, (the actin was labled with pyrene which fluoresces at 365 nm) and then created 4 Arrhenius plots to show that the chamber worked. These results have been incorporated into a sales poster by High-tech scientific.
Week 2: I started of by testing temperature jumps using first pyrene actin and S1 against pyrene actin and S1 (PA-S1 ) going from 20 C to 20 C, this should have given a constant florescence but this didn't happen. So I tried Flouroscein, but again this didn't work. So stumped we moved on to carrying on the previous experiment by increasing the ATP concentrations used, I then plotted these results to find the second order rate constants and then also plotted these against 1000/temperature in Kelvin. I finished the week by completing this.
Week 3 to now: So I started this week by retrying the temperature jumps. This time still using Flouroscein we tested 3 different buffers, the one I had been using was a cacodylate buffer containing 100 mM KCl, the ones I tested where; 100 mM KCl Tris, 100 mM KCl KPi, and 100 mM KCl Imidazole. The best of these was the Imidazole buffer which gave the desired trace and also is affected by temperature compared to the others, giving more accurate results. So I managed to test two temperature jumps one from 20 C to 30 C and the other 20 C to 5 C. These both worked, but my aim was to obtain better results by having the same end point of 5 C, however today everything went wrong as firstly the values on the machine failed meaning the samples were not mixing in the cell so I obtained odd results. We then changed the machine for one that is better suited for low temperatures. So I started setting up my experiment this afternoon only to find another problem that the Mercury/xenon lamp had blown, so I had to waste half an hour for the lamp to warm up. Finally I managed to check this machine was working the same as the other, even though the cell was 20 ul in the first and the new one is 5 ul.
Jonathan
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