Well, I have had quite an eventful week. After little success in viewing any protein we decided to try and reproduce our successful experiment in large quantities and use a new method to view the protein. This will involve solubilising the membranes so that it is easier to apply to the SDS gel, and probably easier for the proteins to transfer to the nitrocellulose paper. However, while we were preparing for this the sequencing for the plasmid we were planning to use came back and it wasn't quite in line, so that was put on hold.
Then yesterday my supervisor decided that seeing as this membrane protein overexpression was proving to be a slow process, he would assign me to a different membrane protein involved in drug resistance while the Masters student continued with the previous one. So, I have been left to plan similar experiments to the last lot, hopefully with more positive results!
Robyn
Wednesday, 30 June 2010
Tuesday, 22 June 2010
Friday, 18 June 2010
The beginning
Two weeks in a lab are over already, and I'm still alive and keeping up! We have already seen some success, so I have decided to blog what's been going on so far.
I am an undergraduate (pre-honours) student at St Andrews University and decided to spend my summer doing something more worthwhile than work in a supermarket as I usually would. I applied for, and thankfully received, a Biochemical Society summer studentship and began work with a Masters student under Dr Law at the beginning of June back home in Northern Ireland. So far it has been much more exciting than being a checkout assistant, and the people are so friendly - definitely worth it!
I spent the first couple of weeks mostly preparing for the weeks ahead. We are trying to heterologously overexpress a membrane protein involved with virulence of Lyme-disease causing bacteria. This involves lots of different broths and agars and other potions, so I was eased into lab work to get stocks of these up. A lot of the work is also centred around Western blotting so I have learned how to set up and run SDS gels and use the very quick SNAP i.d. technique for Western Blotting (which I'm told is fantastic compared to traditional methods, though I wouldn't know). We were able to use this technique to test for our tagged protein in E. Coli.
At first, we weren't having much success for all our hard work. The Western blots showed no protein overexpressed in any conditions, so we set up more experiments to alter the conditions. These were put on pause when Dr Law received the new plasmid, the one that promised to work. This membrane protein is particularly difficult to overexpress in E. Coli, but this new plasmid had shown good results with other membrane proteins. And low-and-behold, on our first try (ignoring a minor hiccup with an SDS gel, oops!), we have some protein expressed!
So the next few weeks are all based around optimising this expression with different temperatures, E. Coli strains, etc. Dr Law has left us to design the rest of the experiments ourselves which is exciting, but I am a little nervous! It is a relief to have another student here to keep me right, I'm sure she has saved my life at some point unknowingly!
I really hope we get positive results next week too, it really makes my day!
Robyn
I am an undergraduate (pre-honours) student at St Andrews University and decided to spend my summer doing something more worthwhile than work in a supermarket as I usually would. I applied for, and thankfully received, a Biochemical Society summer studentship and began work with a Masters student under Dr Law at the beginning of June back home in Northern Ireland. So far it has been much more exciting than being a checkout assistant, and the people are so friendly - definitely worth it!
I spent the first couple of weeks mostly preparing for the weeks ahead. We are trying to heterologously overexpress a membrane protein involved with virulence of Lyme-disease causing bacteria. This involves lots of different broths and agars and other potions, so I was eased into lab work to get stocks of these up. A lot of the work is also centred around Western blotting so I have learned how to set up and run SDS gels and use the very quick SNAP i.d. technique for Western Blotting (which I'm told is fantastic compared to traditional methods, though I wouldn't know). We were able to use this technique to test for our tagged protein in E. Coli.
At first, we weren't having much success for all our hard work. The Western blots showed no protein overexpressed in any conditions, so we set up more experiments to alter the conditions. These were put on pause when Dr Law received the new plasmid, the one that promised to work. This membrane protein is particularly difficult to overexpress in E. Coli, but this new plasmid had shown good results with other membrane proteins. And low-and-behold, on our first try (ignoring a minor hiccup with an SDS gel, oops!), we have some protein expressed!
So the next few weeks are all based around optimising this expression with different temperatures, E. Coli strains, etc. Dr Law has left us to design the rest of the experiments ourselves which is exciting, but I am a little nervous! It is a relief to have another student here to keep me right, I'm sure she has saved my life at some point unknowingly!
I really hope we get positive results next week too, it really makes my day!
Robyn
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