Just as a quick update!
As of 2.41pm Thursday 21st February I am writing my thesis! It's going. Writes itself really...
I wanted to do a quick post about what it means to be a PhD, what you have to do and how you become a doctor. Not a useful doctor mind but heck you've earned that Dr. in front of your name you tell the world!
I know I should have done this in the beginning but hindsight people!
What is a PhD?
You can get a PhD (or Doctorate in Philosophy for those nerds out there) in pretty much anything. If you have the will, the money and a supervisor you get that PhD in the history of snakes in Ireland. The majority of PhDs you'll hear about however will be in science subjects. Maybe some arts subjects (literature etc.) but the effort required to get a doctorate vs the usefulness to your future career means it's typically science-y types doing them. I am very happy to be told about any PhD's readers have done in any subject.
How Long is a PhD?
A PhD usually takes between 3-4 years however that changes wildly from country to country. For example in Scandinavian countries you are required to publish in a peer reviewed journals to be eligible to sit your viva (more on that later). In the UK and Ireland you are not required to publish. In the USA and Canada PhDs can take 5+ years and again some Universities will require you to publish before you get your doctorate. Again I don't know the rules for every country. My PhD is from Imperial College London and they have a max 4 year policy (unless there is some MAJOR need to delay your studies e.g. pregnancy). I am not required to publish. So my PhD is 4 years.
Who pays you?
There are a few ways of paying for your PhD. The usual way is by external funding. This can be from a charity or research council. Applying for funding requires a lot of jumping through hoops and a solid research proposal. The funding process is HIGHLY competitive and a lot of great ideas get left to the wayside. But there's a finite amount of money available and funding bodies need some sort of guarantee that the information generated will be useful for the future. If you feel this is unfair donate to your charity of choice if you know they fund PhDs. This is not an ad but CRUK runs a PhD sponsorship scheme. Another way of doing a PhD is self-funded! Oh yeah. You find the funding yourself or you pay yourself through. I have also heard of supervisors getting funding and they hire PhD students and pay them. It's slightly different from external funding because in external funding, it's awarded to the PhD not the supervisor. Your funding also decides how long your PhD will take. If you have 3.5 years funding you realistically should be finished in that time.
What is the funding used for?
There are three areas your funding goes into:
What does a PhD student do?
I have to say this (and it always gets me) while we may be considered students we ARE NOT students. It's like a job except not 9-5 and definitely not paid well enough. I never had to go to lectures and I never did exams. I could choose to attend lectures (for example I went to some MSc in Epidemiology lectures to learn about a subject I didn't know about). My department also ran seminar series and guest lectures for the whole department but again not mandatory. What I did was work 70+ hours a week in the lab and doing bioinformatics until I got some results I could write into a cohesive story. However I will note I except we are students when it comes to student discounts! You gotta use what you can!
How do you get your "PhD"?
You need to complete a set period of work (3-4 years) which you write into a thesis (in Imperial you do a max of 100,000 words). You then submit this thesis for evaluation and do a viva. A viva is a defence of your work. In my case, I will submit my written thesis to two examiners (an internal and external examiner) who are familiar in my field. They read the thesis and then we come together to discuss the results. I am asked questions and I have to defend my choices. It can take between 2-5 hours to do a viva defence. In other countries your defence is in front of a panel and is open to the public. So you could be standing up in a room full of people (grannys, children, your neighbour) and have to defend your thesis. Usually this happens in countries where you are required to publish. You are already peer reviewed so the defence is more general. One you complete your defence you are given corrections. These can be minor, for example asked to fix typos or a section of your written work, or (god forbid) major corrections, which usually require you to return to your lab/office/hell to re-do work or do more work.
How do I get a PhD?
The best advice I can give you is be open. There are a few ways to go about getting a PhD.
(1) You can pick subject(s) you want to study (I chose epigenetics and cancer) and research all the main people in this field. Contact these people and explain that you would love to do a PhD with them and would they have the ability to come up with a research hypothesis and apply for funding.
(2) Doing an internship/research assistant role in a lab you want to do a PhD in. You will have the ability to show your skills and know when the PI (principle investigator) is applying for funding etc.
(3) Go on www.findaphd.com/ NO JOKE! This is how I did it, I went on Find a PhD and searched my keywords. I found a PhD I liked the look of, got an interview and hey presto I got a PhD. Most labs will try to fill the position internally first (see point 2) but if they can't find a suitable candidate they will advertise publicly.
Is a PhD hard?
Yes. Next question. I joke. A PhD is hard as hell. It's meant to be. But it's also meant to be rewarding and you learn a lot, not just about your subject but about working in that environment and with other people.
What else do I get from a PhD besides a doctorate?
That is completely up to you. You make your PhD what you want. Some people will just do their own work and finish. I however did not do that and I encourage anyone thinking of doing a PhD or doing one to use your time. Teach, whether you do GTA work with undergraduate students or mentoring masters students. Teaching will give you so much in return (not just extra cash - always useful) but also new ways to communicating with others, new ideas and most importantly HELP! I have had three masters students throughout my PhD and if I didn't I wouldn't have the results I have. If you give time and experience to them, they will reward you. Trust me. I also did A LOT of research engagement, probably the most out of any of my peers. I literally said yes to every opportunity that came my way. Why? Because chatting to people about science is AWESOME! I mean come on I'm doing a science blog. It is just so rewarding. I also advise doing courses if you can, I participated in a MSc in epidemiology and did a number of Professional Skills Development Courses (like how to write a literature review, how to make a poster etc.) which really helped my writing and communication skills. As a PhD you are a part of a University or Institution. Use those connections to make the most out of your PhD.
Should I do a PhD?
Honestly I don't know. That is for your to answer. From my experience, if I had been honest with myself I would've probably not done a PhD. I was not and still am not mentally sound (in my opinion) to do a PhD. You require strength and determination. You will be knocked down a lot but getting back up and ploughing on will stand to you. Having said this I don't think anyone would've gotten the results I did or adapt their work the way I did. What you produce is unique and that's worth it. If you want to do a PhD and you really like your subject, do a PhD. Also we need more scientists in the world so if you're a science student wondering if you need to do a PhD PLEASE DO ONE! WE NEED YOU! Did that sound desperate?
What does your PhD mean to you?
I am not sure how to answer that question really. Stupid since I'm the one who asked it. I have a had a lot of struggles throughout my PhD. Struggles which as sadly not unique. I went back on anti-depressants, I had to deal with an eating disorder and I may or may not have tried on numerous occasions to hurt/kill myself. It sounds dramatic but mental health issues in PhDs are not uncommon. You are under a huge amount of stress and have to cope with doing things mostly on your own. This information shouldn't deter you from doing a PhD. I had mental health issues before I started. I may have struggled and I did have to stop my lab work early because I couldn't cope but I also would go back and do it again. I would do it better (obviously) but I would do it again. Doing a PhD has shown me (1) how smart I actually am, (2) what I am passionate about and (3) how strong I am. Plus who doesn't want to say "I'm a doctor" when asked on a plane if there is a doctor on board. Fair enough you do have to add that you in no way are you a medical doctor but god dammit you earned the right to say it!
So that's it! Again if you have any questions please don't hesitate to contact me.
Advances at the Interface Between Metabolism and Epigenetics
16th - 17th January 2019
This year I have the pleasure of attending the "Advances at the Interface Between Metabolism and Epigenetics" conference hosted by the Cambridge Metabolic Network in Cambridge, UK. This two day conference brings together scientists from all backgrounds with two things in common - an interest in metabolism and an interest in epigenetics. Funnily enough these areas interact a lot (which I learnt more about at this conference). When you focus on DNA methylation or histone modifications you forget where these modifications come from. Metabolism as a whole generates small molecules which can contribute to methylation (methyl group) and acetylation (acetyl group) to name a few.
What I have found most interesting about this conference is it's not just focused on cancer. While that's my research area (sort of) I have been to many (many) cancer related conferences. The focus is always "what does this mean to cancer?" whether we are discussing prevention or diagnosis or treatment. At this conference I am learning about the metabolism-epigenetic interaction purely out of interest in these interactions. Now I'm not saying I can't take this information and apply it to my work (which I will be...thanks #EpiMet19) but it's nice to learn something just to learn something.
The conference is two days long and covers a diverse range of sessions including:
Something else I found interesting (and bizarre) was the name "tags". The name tags were electronic and allowed you to "tap" another tag and share information. It also allowed you to vote on the posters you liked (kinda like a nerdy X-Factor). This is the brain child of Blendology (blendology.com/). This is a brand new way of building networks with fellow researchers. A very cool idea however you can't just "tap". It's more of a whack. This is slightly awkward with a stranger you just met!! Also do you really want to say "can I tap you?" or "do you want to tap?" ???
So all in all a really interesting conference.
OH! I presented a poster (again) on my wonderful work. I didn't feel there was a huge amount of interest but sure look! Metformin is an interesting drug with so much potential! If no one is interested it's their loss (this is what I'm telling myself as I weep into my poster because no one likes me! I joke.......sort of). To be fair I was in a bit of a dark area (as you can see in the photos) so maybe that's why.....
Jargon can be a big barrier between people from different career areas. The most obvious barrier for me that jargon creates is between scientists and the wider public but I also find that within the scientific community jargon is still a massive problem. For example when I am chatting with my sister (an incredibly talented doctor) we still find ourselves getting slightly confused about certain words we'll say. We're both technically in the area of biology and medicine but there is still difficulties in communicating.
When you start off in any subject you are given words and definitions and eventually those words just become part of your vocabulary. It becomes so ingrained in your every day that you lose the ability to properly explain what you mean. Honestly try remember the real dictionary definition of a jargon word you use everyday. Hard right? Now imagine you're chatting to someone who has no basis in your area and you're trying to explain the jargon word. Even harder! Why? because every word needs context. And this is where the jargon becomes a barrier. My way of explaining a jargon word ALWAYS ends up in me using more jargon. You end up either getting frustrated and sounding like an idiot OR even worse you end up talking down to the person you're chatting to.
So how do we combat jargon?
Well first thing is to acknowledge you use it.
The next thing is to identify key jargon words you would need to explain and practice how you would easily explain it. Easier said than done.
My scientific explanation:
Cancer cells up-regulate expression of oncogenes while simultaneously down-regulating tumour suppressor genes. Many of these gene expression changes are driver mutations associated with specific cancer types.
Now the tricky part is identifying how much your audience knows before you remove the jargon. You have to ask yourself - do they know what a gene is? Do they know what expression is?
If the answer to those questions is yes then you're in for an easier time:
Cancer cells can change the normal expression of genes by increasing expression of genes that help the cancer cells grow/create energy/create a good environment etc. while at the same time they decrease the expression of genes that would prevent the cancer cells from staying alive for example cancer cells turn off control of their growth so they can grow however much they like. Specific cancer types have gene expression changes in these pro-cancer and anti-cancer genes which can start the normal cells becoming cancer cells. These are called driver mutations because they drive normal cells into cancer.
If the answer is no you have to think harder about what you say:
Cancer happens when normal cells stop acting normal. They start to grow uncontrollably by making loads of energy and stopping themselves from dying. The way a normal cells becomes cancer is different in each cancer type and knowing these differences helps us treat cancer.
The trick here is to not try to explain every word but you completely change your approach. The three paragraphs all mean relatively the same thing.
Combatting jargon requires some practice but once you get the hang of it, it definitely becomes easier and easier. But why is it important? Well for a purely selfish reason it makes you better at writing scientific papers, communicating with your colleagues etc. For a less selfish reason it gives you an opportunity to share your knowledge with people who wouldn't necessarily have access to it. You can help combat the myths surrounding your area such as cancer research (there is not one cure all for cancer that doctors are hiding). You can also help people dealing with cancer understand what is happening to them or their loved one. People want to learn but it's frustrating when you don't know the language.
There are a lot of pros to doing more to combat jargon and being more engaging with the public. Whatever area you're in, give it a go. You might find you actually like the challenge.
As I said before in a previous post a couple of weeks ago (watch-this-space.html) I submitted a blog post to The Cancer Researcher-EACR Science Communication Prize. One of my submissions was shortlisted and has been published on their online magazine.
I wrote this blog post about research engagement and why it's important for scientists to engage with the public. We need to be better at discussing cancer and cancer research with a wider audience. Nearly everyone in the world has been affected by cancer in some way so everyone is interested in learning more. It's also a great way for scientists to be asked questions they have never thought of. Plus it's so rewarding to share what you do on a day to day basis.
Find the link below:
This can also be found on my External Work page:
Recently the European Association for Cancer Research (EACR), who as you know funded me to travel to IARC in Lyon to work, launched a blog competition. The idea of the competition was to write a short blog-style post about "Life in the Lab". You were allowed to submit as many blogs as you liked so I submitted three:
I found out today that the first one (Engaging with the (scary) public – why we need to do it and do it now) was shortlisted as one of the best blogs submitted. So while I didn't win, it will still get published in the next few months.
BUT what I also learnt was that they liked my other two so much that they are going to published anyway probably early next year! How awesome is that! Since they are going to be published I won't be adding them up here but watch this space. Once they become available on the EACR magazine website I will let you all know!
Yesterday I was at the New Scientist Live exhibition in the ExCEL London with CRUK (sorry I am terrible at taking photos so eh no evidence). My job there was to chat to the future scientists from 13 year olds to 23 year olds about how to get a career in research and what it was like to do a PhD. And my one biggest piece of advice was do coding! Bioinformatics is the future. Seriously, even if you hate maths (like me) learning some code language will help you in anything you do.
My other piece of advice was that if you want a PhD google www.findaphd.com/ because it does exactly what it says on the tin. It found me a PhD.
So what is bioinformatics and why is it important? Bioinformatics is basically the analysis of biological data. Biological data usually means sequencing data. You use a coding language such as R, python or unix to analyse this data. The above picture is my code for doing quality control on my RNA sequencing data using unix. I can also (barely) use R. To me python is a snake...
Bioinformatics is important because we can take massive amounts of data from hundreds or thousands of different samples and use this to generate more knowledge about areas such as cancer. This can be sequencing the entire compliment of genes in tumours of hundreds of patients to see if there are common mutations. This can tell us what these tumours have in common and what they don't. You can use this information for example to find "driver mutations" which are mutations commonly found in early stage tumours (p53 mutation is a driver mutation as some cancers require loss of p53 to develop into cancer).
The idea of using whole genome sequencing in cancer has come up a lot recently in the news. And the people who are taking this data and finding interesting results are bioinformaticians. Pretty much every cancer research lab in the world is generating data which needs to be analysed computationally. The data you get collectively from sequencing etc. is called "Big Data". However while most labs are generating this data, a large amount of those labs have no one to analyse it. We need more bioinformaticians to take this data and make it useful. Hence why bioinformatics is the future. The dawn of the era of joint experimental and computational scientists is dawning.
I am an experimental scientist (i.e I could barely use excel let alone do big data analysis when I started my PhD). But I had to learn how to use R and unix to analyse my data because frankly there was no one else. And this learning was something actively encouraged by my supervisor. Every PhD and post doc James has now can do both. It's very important to him. It really should be more important to a lot more supervisors.
So here's my experience: I have done two types of sequencing experiments generating data on DNA methylation (Illumina MethylationEPIC Array) AND gene expression data (RNA-Seq) of two non-cancerous cells treated with metformin. What this gives me in a massive amount of raw data (like hundreds and hundreds of gigabytes - the total amount of data generated from my RNA-Seq was roughly 2-3 terabytes...yeah). I took this raw data and ran it through quality control to make sure every piece of information I got is the best quality. I then ran analyses to determine if there are differences between DNA methylation levels or gene expression levels between your untreated and treated samples. For example if I see a consistent, large reduction in DNA methylation in a certain gene that suggests that there is more gene expression at that gene (low methylation = expression, high methylation = no expression). I can actually overlay this with my RNA sequencing data to see if that gene does actually increase expression! It's pretty cool. I mean you have to validate everything you see in your sequencing data with specific experiments but you can find some very interesting things. So what was the catch (besides learning how to make my computer do this stuff)? It takes time. It took me a full year to analyse my DNA methylation data and validate what I found. I have only started analysing my RNA-Seq data and I can tell you I have sat for 7 hours today watching my computer do aligning on my samples (it's only done 5 out of 9). And god forbid I put a comma in the wrong place or didn't add some arbitrary bit of syntax! I honestly spent a full day trying to figure out why the code I had written didn't work after the computer kept spitting out the "how to" file for the function I had used. I had done exactly what the were saying...except I didn't add two dashes, I had only added one. A bloody dash.
My take home message is the more information we have the more we know. Seems simple right? The gathering of that data is in the whole pretty easy to be honest and a lot cheaper than it once was (though both of my sequencing experiments cost a total of around £9,000 - £10,000 each to get the data). The challenge is analysing that data. Handling terabytes of data to find even one gene shows a change in expression is a mammoth task. We need more people who can pick up a pipette and run an experiment as well as analyse the results of that experiment.
If you are interested in learning any coding language there are lots of resources online as well as courses you can do. My supervisor (James Flanagan) and a fellow bioinformatician (he's a genius - Ed Curry) run a masters in Imperial in cancer informatics. It may seem daunting but if I can learn it so can you. And not just for cancer research or science in general. Knowing how to code could benefit a lot of industries.
Bioinformatics! It's the future people! Sure how are you going to programme your robot in the future if you don't know code??
Resources I found useful:
This year at the Imperial Festival, CRUK wanted to focus on multi-disciplinary research
The second demo was looking at the work the National Physics Laboratory (NPL) are doing on building the "Google Maps of Cancer". The NPL were CRUK Grand Challenge winners giving funding to expand this idea.
The idea is to bring together all the information we have about cancer to go from the whole tumour down to the molecular level. We can use techniques such as Mass Spectrometry to gather information about cancer for example different metabolites produced by cancer compared to normal cells. The demo ask participants to scan the QC code on the blocks. This led to a neat short video about how mass spectrometry worked. At the end you go a result (i.e. the colour the block should be). You turn over the block and leave it. At the end of the day there should be an image from all the blocks turned over.
For more information: http://www.npl.co.uk/grandchallenge/
Time done: 30 months
Time left in lab: 9 months
Time to write up: 9 months
Mood: wildly bouncing from euphoria to depression
So I thought I would do a quick update on how my PhD is going since it’s been a while.
I am officially ok with my hypothesis!
Before Christmas was the hardest my whole PhD has been. I had spent a year analysing and testing my Illumina MethylationEPIC array (which looks at over 850,000 CpG sites across the genome - not that many considering how many there actually are). I found sites that had significant increases and decreases in methylation when I treat with my drug, spent months validating them using pyrosequencing and what did I find? Nothing. It didn’t validate. It was soul crushing.
Buuuut I bounced back. Had a massive rethink with James (my supervisor). We thought about what my drug theoretically does and how that could affect epigenetics and I came up with a new, testable hypothesis. We were assuming my drug does a very specific thing that can be reproduced over and over (eg it always causes the same change in DNA methylation in the same place regardless of what cell line I use or how many times I test it). But maybe it’s not so specific but a bit more random, which is totally fine!
This new direction also means my Illumina MethylationEPIC array analysis wasn’t a waste of time. Celebrations all around!
And it seems to be going well so far. Well except that I have been troubleshooting the same western blot for nearly three months (and beating my head against a wall. Chairs may have nearly been flung through windows). There has been some tears not gonna lie. It’s hard to do the same 2-4 day experiment over and over to prove the result you got in the first place (in bloody January) was correct all along. Science - when you know you’re right but you have to show all the wrong ways to do it to prove you’re right!
So what’s the plan now?
Well I am going to the American Association for Cancer Research (AACR) Conference 2018 Chicago in April (there will be many vlogs, blogs etc) to present what I’ve found so far! Absolutely terrifying since I’ve only started to believe in my results like....last week.
I have about 9 months left of work in the lab before I need to write it all as a thesis. That will consist of learning a few new techniques (RNA-seq, ChIP-PCR) and to get a bit more of an idea of what’s going on and how I can sum this all up in a pretty bow.
I’m also getting another masters student to supervise (YAY) which I’m very excited about because I really like the project I wrote and the results regardless of what comes out are gonna be very interesting. She’s starting at the start of May and finishing up in September.
I need to write some papers! I apparently have to publish my data (who knew) so the next six months will be writing up what I've done so far in a paper, getting it peer reviewed by people in my group so I can find any holes and plug them before December. I can then sent the actual papers off for real peer review and (hopefully) publication.
And that's it. Sounds simple enough but it's taken 7.30am starts working 5-7 days a week to sum up everything I've done since August in a few paragraphs. I've even started drinking coffee in work. I shake violently when I drink coffee. It's not a good idea.
I have one last thing to say to anyone thinking of doing a PhD, to people currently doing PhDs or anyone living with a person doing a PhD:
To the person thinking of doing a PhD: be prepared. I want to scream at you "DON'T DO IT, IT'LL RUIN YOUR LIFE" but I won't. We need more researchers so we need more PhDs. But I will say be prepared. It is not all rainbows and unicorns. It's hard and it will drain the soul out of you. But then you'll get a result and feel like the smartest person in the world. You will love and hate the 3-4 years of your life doing a PhD. But really...be prepared to hate it a lot more of that time than you think.
To the person currently doing their PhD: you are a god damn hero. Keep going. You may feel like you're drowning or that "maybe a masters for this isn't so bad, I should give up". DON'T. You have given your blood, sweat and tears to this thing. You are going to finish. Even if I have to carry you. But seriously. You have got this. You are smarter than you know, you are incredibly dedicated and you will finish. No one knows what doing a PhD feels like until they have done one. No ones knows the soul crushing pain. I know. And I know you have got this.
To the person living with a PhD: please be patient. I know you don't understand what they're doing. I know you don't get why a little comma could cause a massive mental breakdown, the consumption of ALL of the ice cream and you getting shouted at but trust me there's a reason. It's hours, weeks, months or years worth of work that your loved one has just watched go down the drain (this specific example is for programmers but is applicable to all types of PhD). Just keep supporting and loving and caring for the lunatic PhD because they need it. And thank you for being there. Your presence means more than you will ever know.
PS: I know I haven't been posting a lot. I do apologise but every second of my waking life is going into completing my AACR poster so bear with me.
PPS: Apparently I have a very neat desk in my office and all other desks should aspire to look like mine......Weird email sent around the office!? So.....don't get too jealous everyone...
Cute animal photos keeping me sane are courtesy of the wonderful Fota Wildlife Park Ranger (Liam) supporting this lunatic PhD (me).
This is going to be a long and technical one sorry.
This month I want to talk about an experiment I've done pretty much every day for the last three months (plus continuously over the last 2.5 years) - Western Blot.
Western blots are used to visualise and identify individual proteins. In this technique you have a solution (called a lysate) filled with the majority of proteins from cells. This technique separates these proteins out by size and allows you to stain for specific proteins you want (more on this later). This is the standard way to visualise proteins but it is very subjective and is tricky to perfect (I hope you will appreciate why after reading this).
The proteins are visualised by staining them with antibodies specific to that protein and then using a chemical reaction to produce light which can be picked up by a machine. The resulting image is of black bands. The intensity of the black band determines the approximate level of protein present.
There are a few steps to Western Blotting which I will go through. The whole technique can take 1-3 days depending on what proteins you are looking for and what you choose to do in each step.
Steps to Western Blotting:
A little bit of terminology before we begin:
STEP 1: Preparation
Reagents and Buffers
Before you begin a western you need to make sure you have all your reagents prepared. I will NOT be going through this, all I will say is you need reagents for
When you extract your protein from your cells you end up with a protein lysate. This contains the majority of the proteins in your cells. You don't know what the concentration of the total protein is but you are sure that each sample doesn't contain the same concentration. This is due to how well the extraction went in each individual sample but also how many cells there is to extract protein from. There are a few ways of finding out the concentration of your protein which I won't go through (I use the Bradford Assay from BioRad).
What you do need to know is that for each sample you need to make up a solution of protein, water and loading buffer to load onto your gel. You can use as much or as little protein as you want, I typically use 10-15ug per well. I load 20ul per well so I need:
There are many types of loading buffer, I use Laemmli buffer.
The loading buffer has a couple of functions. You are using an electric current to make the proteins travel through the gel. Proteins are all different charges so the buffer makes sure all the proteins have the same charge (negative) - this is done by a chemical called SDS. Proteins are globular structures. These don't run though a gel very well so you need to unravel the proteins into single strands (called denaturation). The buffer will start this process - a chemical called β-mercaptoethanol breaks disulphide bonds. The loading buffer also has a dye in it. This is called the "loading front" which runs faster than your proteins. This allows you to track how far your proteins have travelled through the gel (image later).
A quick note - usually the protein samples you have prepared will be blue once you add the loading buffer and boil it. This is because the dye in the loading buffer is blue. However, the protein I will show in this post will be yellow. This is because I extracted the proteins using acid and the acid in the lysate turns the buffer yellow. Once the gel starts running the dye front goes back to blue.
Gels are made up of:
The acrylamide will set into a gel which has pores in it. The protein will travel through these pores. The percentage of acrylamide used determines how big these pores are. Large proteins run slowly through a gel whereas smaller proteins will run very quickly through the gel. A lower percentage gel (e.g. 7%) will have large pores, allowing larger proteins to run faster through the gel. However you are likely to lose all the smallest proteins. A higher percentage gel (e.g. 12.5%) will have very small pores which slow down the small proteins, allowing you to visualise them easier. However all your largest proteins might not run as they can't past through the pores.
There are two types of gels used in gel electrophoresis - a stacking gel and a resolving gel. They're pretty much made up of the same ingredients (a stacking gel is slightly more acidic pH 6.8).
Steps to making a gel:
I have made up a 12.5% gel for this example to run histone proteins which are approx 15-17kDa in size. I will also stain for a loading control, beta actin, which is 45kDa in size.
STEP 2: Loading and running SDS-PAGE Gel Electrophoresis
The next step is to load your prepared protein into the wells in your stacking gel and running an electric current through the gel to allow the proteins to travel from negative to positive.
The gel(s) are clamped into an electrode chamber and placed in a running tank. The centre is filled with running buffer. The running buffer allows an efficient flow of electrical current to pass through the gels. It also prevents the gels from drying out which you do not want.
STEP 3: Transferring proteins from gel to PVDF membrane
Once the gel has run enough you can stop it. This depends on how long you want to wait for and what proteins you're looking for. My proteins are very small (17kDa) so I can stop my gel once I see the 17kDa ladder band is far enough from the loading front.
The next step is to transfer the proteins from the gel onto a membrane. You cannot stain for your proteins on the gel as it is far to delicate and not permeable - the protein only runs through the gel because we created the wells. The membrane is more permeable and allows you to stain for proteins.
There are a couple of (messy and time consuming) ways to transfer proteins but I prefer to use the iBlot dry technique. This basically transfers the proteins using a machine in 5 minutes (compared to 1-2 hours). The transfer stack consists of:
Again an electric current is used to push the proteins through the gel into the membrane. However the voltage is very low. As we know, small proteins move very quickly. The low voltage prevents the small proteins from passing straight through the membrane.
So how does it work:
After the transfer is complete you need to be VERY fast. You have to quickly remove the stack from the machine, get the membrane out of the stack and place it in TBST. The membrane CANNOT dry out or else all is lost. You can check your transfer at this stage using Ponceau red, which is a red dye that shows up all the wells and bands of proteins but I did not do that today.
STEP 4: Blocking
The next step is to block any unspecific binding. The antibodies are usually polyclonal - meaning they can bind to other proteins that aren't your protein of interest. To prevent this and also to get a nice clean image at the end, you block unspecific binding.
You can block using either of these two reagents:
The reagent you are using depends on the antibodies and proteins you are looking for. I block in 5% BSA/TBST/NaN3.
Before I block I cut my membrane so that I have the two individual gels again. You don't have to do this but I like to. At this point I just cut the excess off using the ladders as a guide. I then mark with a pen on the ladder what gel it is.
The membranes are blocked for 1 hour at room temperature on a rocker.
This is now when we can call out membrane a blot.
After blocking you can cut your blots to stain for multiple proteins. In this case I cut at 36kDa band (blue band above the bottom red band on the ladder) to stain for my histone proteins and also my beta actin loading control.
STEP 5: Antibody Staining of Proteins
This technique as I said uses antibodies which bind to specific proteins and you can them visualise these antibodies using a chemical reaction.
The figure below gives a rough idea of how the technique works.
The antibodies are created in different animal models (please don't ask how cause I have no idea). So if your primary antibody is developed in a rabbit then the secondary antibody will be goat anti-rabbit (i.e an antibody developed in a goat to bind specifically to rabbit-developed antibodies). There are some human antibodies but they're incredibly expensive.
The primary antibodies are made up in the buffer recommended by the manufacturer usually 5% BSA/TBST/NaN3. The recommended concentration is usually given by the manufacturer - typically 1:1000 or 1:2000 (i.e. 1ul antibody in 1ml solvent).
The secondary antibodies are made up in usually either 5% BSA/TBST or 5% Milk/TBST. The BSA doesn't contain NaN3 in the secondary because sodium azide (NaN3) inhibits the secondary antibody. The concentration of the secondary depends on how abundant the protein is and how long you develop it for. For example beta actin, which makes up part of the cytoskeleton of your cells, is an incredibly abundant protein so the secondary concentration is 1:50,000 BUT another protein I look at p-AMPK is not so abundant and the secondary concentration could be as low as 1:1000.
Part 1: Primary antibody incubation and washing
NOTE: for beta actin, you do a 1 hour incubation at room temperature
Part 2: Secondary antibody incubation and washing
For the secondary antibody you can do this at room temperature for 30 minutes - 1 hour, again on a roller.
I make up most of my secondaries in 5% Milk/TBST, except p-AMPK. The casein in the milk removes phosphorylation from proteins so I would lose my p from the p-AMPK if I did the secondary in milk. So for p-AMPK and all phosphorylation proteins I use 5% BSA/TBST.
Again after incubation in the secondary, the blot(s) need to be washed at least 6x5 minutes to remove excess secondary antibody.
The washing part of the procedure is honestly the most time consuming and tedious. It's a lot of back and forth to and from my office desk to the rocker. I usually try to do something else in the lab to not waste the time but sometimes it can't be avoided.
STEP 6: Developing
The final step (thank god) is to develop your blot. This step adds the peroxide and luminol enhancer solutions (collectively known as ECL solutions) to the membranes. You then take the membranes to a chemiluminescence machine which will detect the light emitted and give you an image in return.
You end up with a picture which has black bands. As I said at the start the intensity of the bands indicates how much protein is present - very dark means a lot, very light means very little and nothing means nothing.
The image above shows the loading control (beta actin) for a blot I have done for two cell lines, MCF10A (lane 1-3) and MCF12A (lane 4-6). The bands are very dark indicating that the protein is very abundant. What's important is the bands are almost the exact same size and intensity as each other (different in the two cell lines) indicating that my loading was very even (wahey).
Once the blot is developed you can keep it or you can throw it away. If you keep it and want to stain for another protein or the same protein but with a different secondary concentration you need to strip the blot (i.e. remove all antibodies bound to proteins), re-block using the same blocking buffer and re-incubate with the primary and secondary antibodies. A blot should only really be stripped twice. This will add another day onto your experiment (at least).
And that is it. That is roughly how a western blot works.
The reason why western blots are THE most hated laboratory experiment is because they rarely go the way you want them to, there is always a difference each time you do it and it takes FOREVER.
If a western goes wrong most of the time you have no idea why. It can be anything from one specific buffer which was made up wrong to the wrong gel percentage, the wrong transfer time or transfer voltage etc. Basically everything you did could have an issue associated with it. Trust me I did troubleshooting for TWO MONTHS on one antibody. It eats away at your soul.
Western blotting everyone.
My name is Caitriona and I am a PhD student at Imperial College London, UK.