Nearing the end...

Last week started off with...a bank holiday! So not much science done there, and instead I spent a rainy day at Alton Towers. The rain wasn’t actually that bad, and it meant the park was pretty empty, which was a bonus – only 10 minute queues for Nemesis!

On Tuesday it was back to the lab, and I did a few bits and bobs like transforming and plating out some cells. My supervisor decided he’d like some more of a protein purifying – the HDAC binding partner we’re working on – and so we set about doing that, following much the same protocol as I talked about last week. It went pretty well, with nice thick bands of the protein being seen after gel filtration. There was one new technique we used this time though – there was a contaminating band on the protein gel, and so to further purify our protein, we used ion exchange chromatography. This separates proteins based on their different pI values (the pI is the pH at which a protein has no net charge). It uses a charged column to which the protein of interest can bind, and then buffers of increasing salt concentration are passed down the column to displace and elute proteins – different proteins are eluted at different salt concentrations and hence are separated. I took samples from the wells giving peaks on the trace and ran them on a gel, and then combined the appropriate wells and concentrated down the protein. The gel looked good, with just a single band of our desired protein.

The purification was the major work last week, and since then I’ve also started another scheme of PCR. A while ago I mentioned that I was following a PCR mutagenesis scheme to create HDAC mutants – well, we sent the final product from that off for sequencing, and when it came back unfortunately the sequence wasn’t what we wanted! So, I’m now trying that again, but this time we are going to alter the conditions slightly by using a range of annealing temperatures in some of the rounds to try and improve the output.

I’ve also been doing some catching up on my lab book as it’s the final week! I’ve taken all of the many protein gels I’ve done over the past few weeks, scanned them in to get images, and dried the actual gels down so that I can stick them in my lab book and label them. Drying involves sandwiching the gel between membranes that have been soaked in a glycerol solution and leaving for a while. It’s actually more difficult than it sounds, as you have to avoid getting any air bubbles in the sandwich, which I think I failed at with some of them as this morning I found them cracked into lots of pieces! Never mind, at least I still have nice-looking pictures of them...

So, only 3 more days to go – I’ll give you one final post soon!

Weeks 4, 5 and 6 (a long post!)

Once again, sorry for the big delay between my last post and this one! Just needed to wait for my supervisor to come back to check it over for me.

So, here goes...

Week 4 (seems a very long time ago now!):

This week I did more of some of the stuff I’ve already talked about – I set up some more transfections in tissue culture, transformed and plated out some bacterial cells and started a new scheme of PCR mutagenesis.

The main new things were immunoprecipitation and western blotting, which I’ve learnt about but have never actually done before. We took a batch of cells I had transfected, spun them down and resuspended in lysis buffer, sonicated to aid the lysis, and then spun down again. We then measured the protein concentration with a Bradford assay, and added the correct volume of lysate to FLAG resin (which had been thoroughly washed). This was then left to incubate, the idea being that the anti-FLAG resin will bind to the FLAG-tag on the HDAC binding partner. If the binding partner is able to interact with the HDAC, it will form a complex and the HDAC will also be ‘pulled out’ when we isolate the beads. So, we’re hoping that the mutants don’t interact, or show impaired interaction, with the binding partner, proving the importance of the key mutated residues.

After incubation, the beads are washed several times and they are added to SDS sample buffer for loading onto a protein gel. I discovered that loading samples onto a polyacrylamide gel is not really a skill of mine...I was missing the wells completely at first!

Having run the gel, we then did a Western blot to transfer the proteins to a nitrocellulose membrane for antibody probing. This was another fiddly technique – an air-bubble- free sandwich needs to be made with the gel adjacent to the membrane between blotting paper and foam, and then all clipped into a cassette which can be submerged in the blotting apparatus. The gels we use are pre-made, and very thin and flimsy – I’m a bit scared of touching them as they have a tendency to fall apart!

The transfer was successful, so we then moved on to the antibody probing – we split the membrane to probe different areas with different antibodies. The blots are incubated with primary and then secondary (HRP-conjugated) antibody, with many important rounds of washing in blocking buffer in-between (our blocking buffer is made from milk powder (!) and PBS). It’s a bit strange seeing powdered milk sitting on a shelf in a lab!

Finally, the blot can be visualised with the HRP substrates, and then captured on photographic film  and developed.

The blot was a mixed success really – some of the results were what we expected, others not quite.

Week 5:

This week I finished the new round of PCR mutagenesis I had started – this involved setting up the PCR reactions with appropriate primers and templates, running a sample of the product on the gel to check it had worked, and then using the product as a template for the next round. It was relatively problem-free, with only one round needing to be repeated – I’ve sent the final product off for sequencing now, so we’ll soon see if it worked!

I also helped to set up some HDAC assays to look at the effect of some of the mutations we had made on the enzymatic activity of the HDAC. The assay uses a substrate with an acetylated lysine, and the principle is that the more functional HDAC your sample contains, the more of this substrate will be deacetylated by it. The deacetylated substrate can then react with a developer solution to give a fluorescent product. So, more HDAC activity leads to a higher fluorescence reading. The set-up of the assay involved a bit of maths to work out how much of each component needed to be made up, and then a 96-well plate was used to create the separate drops with different mutant samples. The results we got were quite good – as expected, the mutants showed much lower deacetylase activity than the wild type (indicated by a much lower fluorescence reading obtained by a plate reader).

Week 6 - protein prep:

This week my supervisor was away, and so I worked with one of the PhD students, purifying out some proteins from bacterial cells which we had previously transformed and induced protein expression in. We had 3 different bacterial pellets in the freezer, each of which contained a different expressed protein, and the process of getting the final purified protein samples from these pellets took the whole week.

The proteins were all His tagged, so we followed a basic protocol for purifying out His-tagged proteins. I had to make up lots of buffers first (lysis buffer, wash buffer, elution buffer, dialysis buffer) and then followed the protocol to try and purify out the proteins.

First the cells were lysed by resuspending in lysis buffer and emulsifying, and then the samples were centrifuged to remove debris and the lysate was incubated with a nickel slurry (nickel binds the His-tag on the desired protein). The incubated protein-nickel mixture is then passed down a column and washed with wash buffer several times (lots of watching things drip...) and finally the protein is eluted by adding a buffer containing a high concentration of imidazole (which competes with the tagged protein for binding to the nickel).

A protease was then added to cleave off the His tag, and the sample was then dialysed overnight to remove small molecules such as salts. Setting this up was a bit fiddly – the sample is pipetted into tubing, which needs to be double-clipped at each end to make sure no protein can leak out - I saw the importance of this when one morning I found one of the clips had come off (luckily the second was still in place!). Then the dialysis bag is left spinning gently in the buffer overnight.

Finally, the protein is removed from the dialysis bag and further purified by gel filtration. The principle of this is that proteins travel down a column containing lots of small beads, and small proteins can pass through these beads whereas larger proteins can’t. So, smaller proteins take longer to pass down the column due to taking a longer path through all of the beads, and different sizes of protein therefore come off the column at different times (larger ones first). Glass tubes collect these different fractions, and a trace is obtained which shows you which fractions contain protein. You can then run a gel using samples from the tubes which give peaks on the spectrum to confirm that a particular peak indicates presence of your desired protein.

The whole purification process took about a day and a half per protein, and there were a lot of new techniques for me. So, the PhD student supervised me closely for the first one, but as I kept repeating the techniques I got the hang of it and by the end was doing a lot of it myself. I’ve also got much better at loading the protein gels! (Although there was one slight mishap, where I didn’t add enough buffer and so the current didn’t run properly, with the result of a very interesting looking smiling gel with very distorted lanes...). At the end, we concentrated down all of the proteins by spinning through a concentrator tube containing a membrane which retains proteins above a particular size, and then finally we measured the protein concentration. We found that we had obtained a very large amount of one of the proteins (which we knew we would, as we got massive chunky bands on the gels we’d run) and much smaller amounts of the other 2. From the gels we ran of the products, it looks like this is because only one of the proteins actually expressed properly – unfortunately the other 2 didn’t really express and were full of contaminants. Never mind, we can always try again....

So, that's about it - I'd be surprised if anyone's actually read this far what with the length of this post! Just shows how busy the last few weeks have been....only 2 more to go now!

Sorry for the delay!

Hi everyone,

I realise I've not posted in a while - I've been a bit lazy I'm afraid! And now my supervisor is on holiday, so I can't do a proper post as he needs to check things over before I put them online - so it might be a while before you get my next post, and it will be a long one! Basically, I've been doing some more of the same (more tissue culture, PCR mutagenesis, gels...) and some new stuff - mainly immunoprecipitation and Western blotting. More details to come soon!

Week 3

So…this week I carried on with my rounds of PCR, gel running and gel extraction and finally finished this process, ending up with a final mutant construct that we’ve sent for cloning into a vector.

However, the process wasn’t completely problem-free! In one round I ended up with 2 fairly strong bands in one lane of the gel which was a bit confusing, so I ended up cutting out both of these and trying PCR with both of them as 2 different templates in the next round. This PCR didn’t really work very well and didn’t give particularly conclusive results about which band was the correct template, so we decided that I should run out a sample of the 2 extracted bands on a gel again to look at their sizes. Surprisingly, on this gel, they appeared to be the same size as each other – so I’m not quite sure what was going on the first time with the 2 very separate bands! As both of the bands seemed ok, I took what was originally the more intense one and repeated the PCR with this, which worked.  

Alongside this, I have been helping to purify out DNA from bacterial cultures for transfections. On Tuesday we used a manual method of plasmid prep which aims to give much higher yields than you can get with the ready-made Midiprep kits. However, the price to be paid for this higher yield is a very lengthy protocol! There are many rounds of spinning down for 10 minutes and incubating for 5 minutes, a step of leaving on ice for half an hour, lots of shaking tubes around to resuspend pellets...so it ended up taking the best part of a day to get to the stage where we left the pellet to dry overnight! Still, it’s worth it when you get such a good yield of DNA, as it means you should be left with enough for the rest of your experiments and so saves time later on.

Unfortunately, one of the samples didn’t give us a pellet at all, but we carried on with the other one anyway and picked some colonies for overnight incubation in order to yield some more cells for plasmid prep the next day.

So, the next day I was left to do a Midiprep – a much shortened down version of the manual method, but which yields less DNA. This went well enough – I managed to follow the protocol ok and didn’t make the same mistake as I did last week, which was to snap the end of a syringe off in the precipitator containing the precious DNA! (luckily my supervisor managed to rescue the situation by screwing in a nail and pulling it out)

On Thursday I set up several transfections with some of the cells we had cultured in tissue culture – each one had a different combination of mutant HDAC constructs with its binding partner. This involves incubating the correct amount of DNA (30μg) with the transfection agent, then adding this to cells and media to give an overall 30ml transfection volume – a bit of maths is involved to work out the volume of DNA needed and the dilution factor for the cells. Then each flask needs gassing for 2 and a half minutes before incubation – this can get a bit tedious by the time you reach flask number 6! My transfections actually worked well, meaning my supervisor could use them in immunoprecipitation at the weekend.

On Friday I had another go at the lengthy manual method of plasmid prep with the new pellets we had prepared, hoping that this time it would work. However…it didn’t. I got nearly to the end and things seemed to be going ok, but then unfortunately I lost any sign of a pellet being there. Oh well, I guess that’s just how science is sometimes! Luckily there wasn’t an urgent need for this DNA, so we’ll probably repeat the transformation with a new culture and try again sometime next week – 3^rd time lucky hopefully!

The disappointment of the failed plasmid prep was soon forgotten when we went bowling after work. I hadn’t been for years and had never actually tried it with the barriers down, so I was a bit worried I’d end up with a zero score…but I actually managed to surprise myself and did ok in the first game. By the second game (after a few glasses of wine…) my luck/skill deteriorated a bit! It was all pretty good fun, and a good end to a busy week!

The first 2 weeks

I’ve now completed my 2^nd week of the placement, and so far it’s going pretty well – I haven’t managed to destroy anything yet!

My first impressions of the lab were very good – I was given a tour and saw how well equipped it is, and I was introduced to the other group members who are all very friendly. There is a good mixture of PhD students, postdocs and a couple of other summer students, and in total the research group has around 11 members – which I’m told is quite a lot!

My project is to work alongside my supervisor, doing some experiments looking at the interaction between a histone deacetylase (HDAC) and its binding partner. It took me a while to get my head around the project – the research in the lab centres around the structural biology of transcriptional corepressor complexes, which recruit and activate histone deacetylases to repress transcription of target genes through modification of chromatin structure. My role in the project is to help make mutations in the HDAC in previously identified key residues and regions of the protein thought to be essential for interactions with other components of the transcriptional corepressor complex. We can then look at the effect of these mutations on the binding and activity of the HDAC in order to confirm the importance of these residues and regions.

Although I had some prior knowledge about transcriptional repression and histone deacetylation, I hadn’t before looked in detail at the crystal structures of transcriptional corepressor complexes, and so I had to do a fair bit of paper reading to get my head around it all! The first week involved me being introduced to a lot of new techniques – I learnt how to do a MidiPrep to purify out plasmid DNA, how to make and run an agarose gel, how to set up and run PCR, how to do gel extraction – all things I had learnt about and seen bits of in practicals, but never done on my own. I also learnt the principles of mammalian tissue culture, which was completely new to me and quite interesting. I must admit I felt a bit useless for the first few days, not knowing where things were in the lab, having to constantly ask for help and keep having things repeated to me...but luckily my supervisor was very patient and helpful, and by the second week I was getting the hang of things and felt a bit more confident!

In the second week I was able to do much more on my own – I was left to introduce two mutations into a construct using several rounds of PCR with mutant primers, and this involved setting up the PCR, running a gel to confirm it had worked and then extracting the DNA from the band to use again in the next round. I was always surprised when the PCR actually worked! The process gets quite repetitive, but it’s all good practice and I’m getting faster at doing it each time.   

I also had a go at some tissue culture – we had to grow and subculture mammalian HEK293 cells in preparation for transfection of our mutant constructs. Using sterile technique and working under a hood is vital in this process to prevent contamination of the cells, and I found that it really takes quite a lot of concentration to make sure everything remains sterile – you can’t go waving the pipette around and leaving lids off things, and everything has to be copiously sprayed with ethanol. So far my cells have stayed alive, which is promising!

All in all, I’ve had a very enjoyable first 2 weeks and feel well settled in to the lab now – I’ll keep you updated on how it goes!