Friday, 25 July 2014

Long time no post...

So I haven't posted about what I've been getting up to in a while, this is because its been a bit of a slow week. After running out of enzyme at the end of last week, I've been waiting on the delivery to come all week with it finally arriving this morning! So I was quick off the mark to get some more assays running to try and get a result.
The gel of the results is running as we speak and I'm also having a quick session on the EM later this morning. So after a slow week, today should be pretty busy!

Also, I am reaching the end of my internship, with having only one full week left. I can't believe how fast the time has flown! 7 weeks gone already!! Know we just have to figure out what to do next to finish off my project, write the report and the jobs a good one! haha.

Wednesday, 16 July 2014

It's working!!!

I finally got my column working yesterday, after about a week of trying different things to clean it out! I have no idea what happened to it, or what made it start working again, but all I need to know is that it is in fact again working!

And I have my best result yet for dissociation of TCV!! The graph I obtained is very similar to that of the paper I am working from (mentioned in an earlier blog). Let's have a look shall we!! (if you can't tell I'm pretty happy about this!)

This is the image from the original paper, the graph shows the absorbance at 280 nm. The gel underneath corresponds to the fractions taken at each point on the graph.

And this is my graph!! The is a plot of both absorbance at 260 and 280 nm on my graph. The two peaks can clearly be seen and they are of good resolution and smooth (unlike some of my other attempts!).
The first peak corresponds to the rp-complex of the TCV capsids and the second peak corresponds to coat protein.
I ran fractions from both peaks and they were both concentrated enough to show up on the gel! Which is another first!! So I'm pretty happy with yesterday's successes all in all!!

Monday, 14 July 2014

Battle 2 with the Electron Microscope.....

My second battle with the electron microscope took place last Thursday, and I can happily say that it was a successful one!!

I tested a few different staining techniques as my last images would not focus because of the techniques used for staining.

My first image was staining with uracil acetate 3 times but blotting straight away. These were the results...

Much better than the previous trail, as you can now see some of the more detailed features of the virus and see that it is in fact hexagonal. However the background is pretty salty! But better was still to come!

My second staining method was using uracil acetate but leaving the sample to stain for 30 seconds, unfortunately this method didn't give any images that were in focus so it was concluded that this staining method was not successful.

The third method I tried was leaving the stain for 1 minute. Here are the images...

 I think these are the best images of TCV that I took, and the most successful staining technique!

When I left the stain for 2 mins, the sample was slightly over stained, but still in focus!

Some of the detail can still be seen, but the virus capsids appear a little bright in contrast to the background, indicative of over staining. 

So I have found out that the best staining method, for me anyway (there are loads of different protocols that say otherwise with different methods), is to leave the uracil acetate on the grid for 1 min before blotting off the excess. 

All in all a pretty successful ending to a unsuccessful week!  

Thursday, 10 July 2014

The route of the problem!

I think that I may have solved the mystery of the disappearing virus! YAY!!

First thing on Wednesday morning we decided to troubleshoot the column I have been using to separate my dissociated virus as a lot of my sample was getting "lost" somewhere. After the first test it was clear that the column was not working how it was suppose to and the pressure was way to high!
So I cleaned the column with harsh chemicals to get rid of anything that was blocking the column. Then left the column in run with water over night to get rid of all the chemicals we had previously put down it.

This morning when I returned nice and early, I looked at the chomatograph of the column and saw that lots of "stuff" had been eluting over night!! In fact it's still eluting as I type now! So there was a lot of contaminating molecules stuck in my column, hopefully explaining my poor yields!

So the only thing I can do right now is wait for the elution to finish and then try again with my dissociation! As they say, if at first you don't succeed, try and try again! Fingers crossed this time!!

Tuesday, 8 July 2014

Why won't you concentrate?!?!

So, a pretty unsuccessful beginning to week 5 of my internship.

I am still trying to do my chymotrypsin digestion experiment, but I am having trouble getting my sample concentrated enough to show up on my SDS PAGE gels! I am losing a lot of sample when I inject it onto the column, I don't know why, it's just not coming out! It's a little frustrating as I know that I can do the experiments and understand it, but I just can't get the result I want because of one small detail! I have heard from the other post docs and PhD students in the lab that this happens regularly in research. Of course I didn't expect everything to go swimmingly over my 8 weeks, but it would be nice for this experiment to start working sometime soon!

But all I can do is keep trying! I will keep you posted!

Friday, 4 July 2014

End of week 4

So its the end of my third week here in the Stockley lab, and its been a busy one!
Alongside moving house, I've been trying to complete my protease digestion experiment (mentioned in a previous post), which has proven to be more difficult then I first expected.

The protocol I am following is pretty straight forward, but with my first trail I encountered some problems. All my samples looked the same when analysed by SDS-PAGE, which meant possibly all my TCV sample was being digested by chymotrypsin as soon as I mixed the sample with the enzyme.  This is not what I expected or wanted to see!

Time to troubleshoot!!

First I tried to lower the amount of enzyme I added to the assay. No success.
Then I thought maybe my protein has degraded and that's why I'm not getting a result? So I tested for that on a gel alongside chymotrypsin on its own.
Conclusion? I think my protein is of very low concentration and can't by visualised by now I have to figure out where to go from here, especially as the amount of sample I have left is very little!

So this week hasn't had any major discoveries of science unfortunately, but that's okay because sometimes it takes a while for an assay to work! Also if I do a PhD, this is good practise for me, getting used to the fact that not everything in the lab will go right first time and sometimes you have to work on the same experiment for a while before getting a result! All experiences of what a real career in the lab will be like for the future!