Honest Protocols: SDS PAGE

So, this time we looking at the fun and active (full sarcasm intended) stage of the western blot: SDS Page Electrophoresis. This is a form of gel electrophoresis specifically for proteins. When I had to do this for the first time, I really struggled to find any practical information on how to actually do it and was so reliant on the kindness of lab members to help me out. Still took me ages to do it probably. So, I'm hoping this one will be helpful to any poor person who is trying to do this for the first time. 

The point of the SDS electrophoresis is to separate molecules like proteins according to their size. Because the proteins have a charge, an electrical current separates them out. The smaller molecules move faster and further than the heavier ones. This allows you to see what proteins are actually present in the sample. Each protein forms an isolated band which you can work out the size off.  You can do further experiments with these separated proteins -such as the western blot. 

What a SDS electrophoresis looks like. 

So down to business:

1) Draw up the excel sheet where you calculated how much of the sample you actually needed for the electrophoresis*

*The amount of protein you need for a gel electrophoresis is about 10-50 µg per sample. If you have too much protein, it won't migrate probably. So, what you need to do is dilute the samples down with some distilled water. You don't want to use the entire sample so the best way to do is work out the concentration of the sample, work out how much µl you actually want and add the required amount of distilled water to get the amount of protein down to 10-50 µg. So, for example, if your BCA tells you have 20 ug of protein and you took 10 µl of lysate for the BCA, that gives you a concentration of 2 µg/µl- which is also 2 mg/ml. So, for every 10 µl of lysate, you have 20 µg of protein. Now let’s say you want 10 µg of protein per well. But let’s say the gel well holds up to 40 µl of solution in total. You decide to make it out to 30 µl so you need 5 µl of the lysate plus 25 µl of distilled water. 

2) Hunt down the distilled water and the Laemmli buffer* from wherever the last person put it.

*The Laemmli buffer is used to give you better bands as it helps with the isolation of the proteins. It has five crucial components including a dye, but most labs have it already made for you. Fair warning, it smells AWFUL. When adding it to your water + protein mixture, you want the L buffer to be a sixth of the total volume- including the buffer. So, if the protein and the water combined are 30 µl, you can add 6 µl of the L. buffer. 

Laemmli Sample Buffer from Bio-Rad. It's purple and smells gross. 

3) Sulk at the smell of the buffer. Question your life choices.         

4) Consider the gel percentage* you will need.  A

*Gels are made out of something called polyacrylamide which gives you a matrix full of pores. The proteins move though the pores to separate out. Proteins between 25-100 kDa will need about 8% polyacrylamide but a smaller one of between 4-40 % kDa will need about 20 %. More polyacrylamide will give you decreased pore size. So larger pore sizes are better for your bigger proteins. Of course, if you are trying to investigate several proteins that have different sizes, you may have to run them all on separate gels which is just a nightmare. You can also use something called a gradient gel which is made up with a range of concentrations. 

5) Hunt down the helpfully pre-cast gels* in the cold room. 

*Pre cast gels are a luxury that some labs get. The gel is already made up for you, and it is contained within a plastic casing. It also has been created with a plastic comb which creates the wells you will need. The wells are where the samples are going to go. 

A precast gel with green comb and tape in a wrapper. 

A gel after running the SDS page. The gel itself is contained within the plastic 

and the blue is the L.buffer that has travelled down. 

6) Remove the comb and the sticky tape* from the gels.

*The sticky tape is designed to present the gel getting dehydrated or breaking in transport.  But you don't remove it, you stop the electrical current from getting to the gel. So, for the love of GOD, remove it!

7) Place gel in the tank*. Realise you are going to need to get a second gel and run two gel electrophoresis*.

*The tank is what holds the gel and has the running buffer which is going to carry the electric field. It also has two electrodes - a positive anode at the bottom and the negative cathode at the top. The proteins are going to move down towards the positive anode as they are negatively charged. The lid is also incredibly important and is going to connect the tank to your power supply. 

                                         An SDS PAGE tank with the lid. 

*Each tank holds two cassettes which holds two gels each. So, you get four in total.  You only need to use one cassette - but to seal the cassette probably to minimise leaking, you need to use two gels. You could potentially use a blank which is just a piece of plastic the same size as the gel and its plastic casing, but they are always a nightmare to hunt down. So, it's just easier to run two. 

Tank showing the cassette. The gels get clamped in front and behind of the white plastic

 and are held in place by the green. 

8) Make up a second set of samples for your second gel electrophoresis. 

9) Hunt down a power pack. Remind yourself how to set up a tank*.

Power pack. It's usually lurking on someone's lab bench. 

*Luckily these tanks are usually colour coded. Just remember: RED ELECTRODE TO RED SIDE

10) Realise you have run out of running buffer*

*Running buffer is what the electrical current is going to travel though in your SDS PAGE electrophoresis. It's made up of Tris - which keeps the pH stable, glycine which helps the proteins line up neatly before separating - think of it like them all meeting at the same starting point, and it also has SDS. This keeps the proteins unfolded and negatively charged.

11) Make up some more.  Make up extra to use in another electrophoresis in a rare exhibition of forward thinking*

* Because you use so much of it, you're better off making a 1 litre stock that is 10 X more concentrated that you need and then dilute it 10 times when you are using it. For example, take 100 ml of your stock and dilute it with 900 ml of running buffer. 

12) Fill tank with running buffer*.

*You want enough to cover the wells in the inner chamber - the space between where the two gels are clamped in the cassette, and enough to cover the bottom edge of the gel in the outer chamber. It's about a litre.

You can just see the running buffer in the outer chamber, and in the inner chamber, 

it should be filled to the top of the wells. 

13) For once, remember to wash out the wells with running buffer*

*Sometimes the wells get clogged with the gel, so you want to make sure they are empty. 

14) Scrabble blindly in the freezer and eventually hunt down the ladder *

*The ladder is a mixture of proteins with known molecular weights. It's like a size ruler. You can compare the bands that your sample produces with the bands the ladder gives you to estimate the size of the proteins you have. 

A ladder produces something like this. Each ladder comes with a guide 

telling you what size each band is.

15) Mentally brace yourself for the fun of loading* into the well.

*Loading is almost as hellish as a western blot. You are using a pipette to place small amounts of fluid in a well in the gel which is almost invisible. 

Like that. It's easier once you have the first well filled. 

16) Load 4 µl of ladder into each well. Decide to use the ladder to help you distinguish between the left and right of the gel.  Load two ladders at the left side, one at the right*

    * This will be important as it will help you remember what samples were actually in each well. The gel will be flipped around later so it's hard to remember which well was which. But if well 1 was the one next to two ladders, you can work it out. 

17) By some miracle, you manage to get the ladder into the well without missing it or stabbing the bottom of the well with the pipette tip.

18) Brace yourself for more loading.

19) Load 13ul of your prepared samples into the wells. Cry internally.

20) Mentally swear abuse at whoever is making noise in the other end of the lab.

21) Breathe for the first time in five minutes when you finish without stabbing any wells or missing any. 

22) Put the lid of the power pack on your gels. Remind yourself again that it is RED to RED. 

23) Attempt to run the power pack.

24) Scream when you see there's an ERROR alert on the powerpack.

25) Realise it's because your tank is leaking and the running buffer is no longer covering the tops of the well*.

*Leaking tanks are a rite of passage. You will spend ages trying to find the one tank in the lab that doesn't leak, think you have found it and not realise you have not got it until it's too late. 

26) Clean up the buffer that has leaked over your table and top up the tank.

27) FINALLY, the powerpack is good to go.

28) Settle on a voltage of 100 V *.

*100 V is a standard voltage to use. It's a good one to use if you haven't mucked around with the protein before. 

29) Debate whether you should run it for 1 hour or 2 hours.

30) Decide on 1 hour and you will come and check on it later.

31) Go grab a coffee and hunt down your will to live. 

32) Come back to the lab and decide it still needs a half hour.

                                     The samples should form a purple line and

                                             gradually move down the gel.

33) Hike up the voltage to 120 V.

34) Decide you can't be bothered to take off your lab coat again so settle down to wait. 

It looks a bit like this when it's finished. Either side is the ladder.

 You can only see the dye in the samples but proteins of larger sizes will be present

 further up the gel. The next stage is to do the western blot to see what exactly is present.  

 Once the gel has finished, it's time for transferring and blocking which will be explained in the next honest protocol.  Which is more hellish than the loading. To be honest, everything gets hellish from this point on. HAVE FUN!