Honest Protocols: Transferring and blocking

 

Honest protocol: transferring and blocking!

This is my latest post in my honest protocols series. At this point, we have finished with the electrophoresis and can now move onto the biggest hell that any scientist will ever have to face... the western blot!

 The point of the western blot is to see what antibodies are able to bind to the proteins that have separated out on the gel. I'll explain a bit more about what the antibodies do and how this helps us identify what proteins are present next time, but what we need to do is get those proteins from the gel onto the western blot membrane. This membrane is a thin porous sheet that immobilise the proteins. Once the proteins are on the membrane, you can start using antibodies to see what proteins you actually had in the lysate in the first place.  The real problem with westerns blots is that you can't see if it worked or didn't work until the last possible moment.  At this point, the electricity for your electrophoresis should have been switched off. The gel can sit in the tank quite happily whilst you get the next stage sorted. 

1) First, decide what membrane you actually want to use*. If using PVDF, soak it in methanol for 5 minutes. Grab the methanol from the hazard cupboard, start to pour it out.... then realise that you should really be doing this in the chemical fume hood. Stop yourself before gassing out the entire lab and sheepishly shuffle to the fume hood. 

*Sometimes, you don't actually get a choice. Your lab stocks one kind and that's that. There’re two types of membrane: nitrocellulose and PVDF membranes. Both have their advantages. Nitrocellulose is better for small proteins but insanely fragile. PVDF is better for your high molecular weight proteins and is also less fragile. You have to soak it in methanol before using it because it is hydrophobic and won't work otherwise. Nitrocellulose can be used instantly. 

2) Retrieve the stacks* from the random-looking cupboard they are usually left in, after spending about 10 minutes trying to work out where they are. 

*Stacks are basically a 'stack' of filter paper. It's designed to be thick. Some labs will just buy thick filter paper, others will buy standard filter paper, and you use stack layers of it together to make a 'stack'. There's always some floating around. If you're lucky, some kind and organised person will have cut it into nice convenient sizes.  Other times, you spend another 10 minutes trying to hunt down thick enough scissors. 

Literally just thick filter paper. 

3) Hunt down your pre-made transfer buffer* that you made in a rare display of forward thinking.

*This is the conductive solution that moves the separated proteins from the gel onto the membrane.  It's a buffer agent - usually Tris, to maintain the pH combined with some methanol to get the protein transferring to the membrane and a little bit of SDS which helps move your large proteins. Sometimes it arrives pre-made which in theory makes your life easier. 

4) Discover that someone has used your transfer buffer. Be angry for a few minutes and then remember that your lab bench neighbour asked if she could and you were generous enough to say yes.

5) Regret your generosity for five minutes.

6) See that she has made some up. Pop your head around the cold-room door, see that she is in the lab, and yell out across the lab to get her attention. 

7) Ask if you can use her buffer. Receive answer in the affirmative*.

*If the answer is in the negative, odds are there is another helpful person who will let you use theirs. Or will help you make some of your own up. 

8) Pour transfer buffer over your stacks and leave them to it whilst you hunt down a transfer cassette*

*The transfer cassette is what you seal your gel, membrane and the stacks in. It stops the gel and membrane from moving about, keeps them in constant tight constant and applies a constant electric field that pushes the proteins onto the membranes.  

A transfer cassette with the lid on the left. 

9) Start assembling the transfer sandwich *. Lay a stack down on the cassette and place the membrane on top.  DO NOT TOUCH THE MEMBRANE*.

*This is actually what it is called. 

*Otherwise, the protein on your fingertips gets transferred too and you end up with a massive fingerprint. 

10) Hunt down a roller* and run it over your transfer half- sandwich.

Roller. 

*It looks like a mini version of those rollers you use in painting walls. The point of it is to get rid of any air bubbles. They get in the way of the electric field and stop the protein transferring. 

What happens when you don't roll.. 

11) Fish out gel and it's plastic casing from its tank. Decide that you only used this buffer once and you can't be bothered to make up another load for your next western blot* Pour it back into its bottle, only spilling a quarter of it in the process.

*You can use a running buffer two or three times. No more than that. Some don't bother and just make a new one. 

12) Crack open the gel from its plastic casing using the handy cracking tool that some kind lab member lets everyone use and the arrows on the plastic surrounding the gel. Pray to any god you do or do not believe in that the gel will not break in the process. 

Handy cracking tool. It probably has a proper name. 

13) Somehow place the gel neatly on top of the membrane *.

*Easier said than done. The way I used to do it was use the cracking tool to take off half of the plastic casing. The gel should be lying on top of the other half. From there, you have two options. You can gently lift it up by its corners using plastic spatulas and lay it on the membrane. Or, you can flip the plastic casing over so that the gel is facing the membrane and use the spatula to slowly nudge it off the plastic and onto the membrane bit by bit. At some point in your western blot life, the gel WILL break and you will cry. But you might still get some data.  Put the broken pieces on the membrane and try to piece it together.  This is also why we used the ladder to denote each side of the gel.  If you flipped the gel + plastic, or the gel at any point, it can be hard to work out what well actually had what sample. You put sample 1 in well 1 but is well 1 now on the left or the right? If you did two ladders on the left hand side, you know well 1 is always the one next to the two ladders. 

The plastic spatula things- 

these are actually known as gel releasers. 

14) Roll it out again and place another stack on top of the gel*

*The reason you do it this way with the gel on top of the membrane is because the electrical field that the transfer cassette is creating acts downwards. The top of the transfer cassette will be negative whilst the bottom of the cassette will be positive.  Proteins are negative so they will move away from anything negative but move towards anything positive. 

Handy diagram of the transfer sandwich.

15) Pour some transfer buffer on top.

16) In one swift clean motion, slam* the top of the transfer cassette on top of the sandwich and lock it in place by turning the dial. 

   *Okay, you don't actually have to slam it. But you don't want the lid of the transfer buffer to dislodge any part of the sandwich and add air bubbles. You can't just slide it in place or shuffle it about. It has to be placed in exactly the right location in one motion.  

17) Blindly panic that you did the transfer sandwich wrong as you can't remember what you actually did.

18) Wonder if you should open it up and check... but you also don't want to have to reassemble and get the roller out again as you are also scared that you will break the gel.

19) Shove the cassette in the machine. Wonder what voltage you should use and for long*.

*It depends on the size of the protein.  You want the proteins to move off the gel and onto the membrane but if you leave it too long, the proteins can move though the membranes and into the stacks which you don't want. Large proteins need longer time and potentially more voltage Honestly at first, it's a bit of trial and error and see what happens.

Transfer machine with two cassettes.

20) Decide on 10 minutes and 25 V*.

*This is a standard reasonable time. Some transfer machines don't give you the option of choosing voltage but say things like 'moderate weight', 'large weight' and 'mixed weight'. Problem is, they don't say what counts as 'large weight'. 

21) After 10 minutes, use tweezers to lift up the corner of the gel. If the ladder has moved off the gel and onto the membrane*, it's done. 

*You can see the ladder on the gel when the electrophoresis is done before of the dyes in it. When the transfer is done, there should be nothing left of the ladder on the gel, and you should instead see it on the membrane. 

22) Cry with absolute relief. You didn't want to slot it back in the machine for two minutes intervals and check in on it each time as it's flipping boring and irritating. You also think you moved the gel when checking on it*.

   *If you move the gel before the transfer is done and then put it back, unless you put it back in the EXACT same spot, you end up with double proteins transfer -two overlapping protein bands when you should only have one band.

Double protein transfer. 

23) Dissemble your transfer sandwich. Start to throw away your stacks then remember that your lab is supposed to be eco-friendly and you can re-use them.  Deposit them in some plastic tray somewhere on your bench to dry off. Discard the gel * after squeezing it and playing with it for a few seconds, enjoying the sensation. 

*After all that work, it’s now useless to you.

You membrane should look a little like this at this stage.

All you can see is the ladder and none of the protein 

from the lysates. 

24) Decide that you are too lazy to continue with this and decide to block * the membrane overnight.

*Blocking is what you do to your membrane to make sure only the proteins in your lysates actually bind to the antibodies you will apply later.  It's basically covering any part of the membrane that doesn't have protein on it with unreactive proteins. They won't bind to any antibodies.  You can either do it for one or two hours at room temperature or just leave it overnight in the cold room.  Like all stages of the western blot, you will not know if it worked until the absolute last moment. 

25) Hunt down your blocking buffer * from the fridge. 

*You're in a fancy lab so you are using Bovine Serum Albumin. Milk also works so some labs just have milk powder which you make up with distilled water.

26) Grab your plastic western blot membrane holder thing*

*Any plastic thing with a lid works well. There's probably some lab supply company selling official western blot blocker holders, but you can use almost anything. 

It's like an old video cassette. Think they are officially 

called 'blot trays'. But anything with a lid works. 

27) Pour the blocking buffer all over your membrane in the plastic holder thing.

28) Wander over with the plastic holder + membrane + buffer over to the cold room and find some room on the shaker *.

*The shaker keeps the membrane moving at a steady speed. It's making sure that the blocking buffer is going to reach every part of the membrane evenly.  

Like this. 

29) Check that the membrane is moving independently inside of the plastic holder*.

   *It should be moving with the shaker and not staying still. Add more buffer or use a different holder if it’s not.

30) Call it a day there and go hunt down a coffee. Fortunately for you, as you decided not to block in 1 hour, you make it to the cafe just before they close.  Go find a friend to vent about your western blot horrors in an attempt to find sympathy.