Honest Protocols! BCA assay

 Third entry in my honest protocols series and today we are talking though the BCA assay.

Once we have collected our lysates, we need to know how much protein is actually contained with the lysate.  This is so we can actually use these samples for experiments. So, we use something called the BCA. I always hated having to do this, because I just couldn't win either way. If the assay didn't work, then my samples didn't have the protein I needed and they were useless. But if the assay worked, then I would have to do a western blot. Which was just absolute hell. But the one consolation is that it was reasonably quick to do, giving you some time to accept your fate and you get a nice purple colour when the assay works. 

The purple solutions.

The BCA measures the protein concentration using something called the Biuret reaction. In this reaction, you add a base to your protein making it alkali. Then, you add copper sulphate. Copper sulphate has copper ions that have a charge of 2+. Peptide bonds - the bonds holding amino acids together to form a protein, have nitrogen ions. These nitrogen ions react with the 2+ copper ions and make them 1+ copper ions.  By detecting how much of these single charge ions you have, you can in theory work out how much protein you have.

The best way to work out how many 1+ copper ions you have to create a colour change. This is where the BCA comes in. BCA stands for bicinchoninic acid, and it reacts with 1+ copper ions to form these great purple colour. So, the more purple you have, the more protein you have. 

What you also have is are the Bovine Serum Albumin standards -or BSA for short. These standards contain a known amount of protein, so you run the assay, and create what's called the 'standard curve'.  You can then run your actual samples and find the data on the standard curve, telling you how much protein you have in your samples. 

The BCA assay kit I used always contained a few bottles. The BCA held in an alkaline environment which was always in this big bottle, the copper solution which was in a little bottle and is bright blue and the Bovine Serum Albumin (BSA) standard which were in these tiny ampules. So, I used to remember them as the Big Bottle, the blue stuff and the tiny bottles.

The big, the tiny and the blue.

The copper sulphate solution has to be added to the BCA solution just before you use it. This is because adding copper sulphate to this solution makes a very chemically unstable solution that starts to degrade pretty quickly. So, it stops working if it gets too old. Bit ageist to be honest. 

Anyway, here's what you need to do in as much detail and as honesty as I can possibly give:

1) Hunt down the BCA box that's somewhere in the lab.

2)  Realise it is not in its usual location.

3) Panic for a moment, thinking that there's no BSA anywhere in the lab and you won't be able to run a western blot. Brighten up for a moment realising that you won't HAVE to run a western blot. Then sulkily realise that Kate at the lab bench across the room was also doing a western blot and nicked the BSA, and you no longer have an excuse to get you out of having to do the western blot.

4) Stomp across the lab, have a nice conversation with Kate and walk back with the BSA.

5) Consider how much of the big bottle you will need and how much of the blue stuff you will need to add*

*It's 0.2 ml of the blue stuff to 9.8ml of the stuff in the big bottle.

6) Realise you haven't even got your 96 well plate yet, so wander over to the store room to retrieve it.

7) Your favourite lab marker pen* ran out yesterday and you haven't replaced it so yet, so wander to annoy your favourite lab friend and wheedle a lab marker pen out of them.

*Alongside blue roll, lab marker pens are one of the most useful things ever. These pens are designed to withstand the harshest temperatures and the only way you are getting it off your lab equipment is a lot of ethanol and elbow grease. 

8) Label your 96 well plate CLEARLY*

*An unlabelled 96 well plate can ruin everything. You need to mark where your standards are going to be and where your ACTUAL samples are going to be placed, along with your controls.  

Diagrams like this are useful for planning

 where each of your standards and samples will go. 96 well plates have a lid so you can use your lab marker to actually mark the lid where everything should go. 

9) Make the BCA, put it aside and start making up your BSA standards. 

10) Realise that the standard is in this flipping glass ampule that you have to break open to access the liquid. 

Like this. The join is always weaker where that green band is

 so that's where you should aim to break it.

11) Gingerly break it open without cutting your fingers. Always a miracle. 

12) Create first standard with 30 µl* distilled water and 0 µl BSA. Do not create it directly in the 96 well plate. Make it up in E-tubes.

*µl stands for microlitre. It's a flipping tiny measurement.  

13) Create second standard with 24 µl water and 6 µl BSA. 

14) Keep going until last sample contains 0 µl water and 30 µl BSA*

    *If this sample goes purple, you have majorly screwed up. This sample should not contain any protein. 

15) Vortex all your samples for a few seconds or so. Stick your finger in the vortex when no one is looking.

 

Standard Lab vortex.

16) Pipette your standards onto your well plate. Each well should have 10 µl and you want to have at least two wells for each standard. Three is better. 

17) Panic for a minute when you forget which one of your E.tubes had which standard and there's a chance you pipetted the wrong one into the wrong well. Decide you can't have because there's still the same number of liquid in the E.tube that shouldn't have been used as all the other tubes. The only one with less liquid is the one you should be using. 

18) Realise it might have helped if you labelled the E.tubes with the coordinates of the relevant well but that no one is ever that forward thinking.   Decide you will do that next time, knowing that you will have forgotten by then.

19) Hunt down your lysates in the -80 °C freezer. 

20) Realise that it might have been useful if you had taken them out of the freezer before you started wandering around the lab, because now you need to wait for them to defrost.

21) Luckily, the samples are small enough, and shouldn't take too long. You half -heartedly flick them*

*This actually works to defrost small volumes. 

22) Your samples finally defrost so you can start pipetting them. You want 1 µl of sample and 9 µl of water, and you want to do this for three wells.  Shove samples back in freezer- unless you are planning on actually doing the next stage of the western today. In that case, shove them in a load of ice in an icebox and leave them on your lab bench.

23) Add 200 µl BCA assay to filled wells.

24) Add 210 µl BCA assay to three empty wells - these are your controls. Realise you didn't label them on the 96 well plate. Quickly do so with your lab marker.  

25) Take your plate to the hot plate storage thing that you still don't know the actual name off and incubate for half an hour at 37 °C. 

It looks like this. 

26) Go amuse yourself for half an hour. Might as well get a coffee and something chocolately to console yourself about having to do a western blot. 

27) Retrieve your plate and head to the spectrophotometer*

*Spectrophotometer is a piece of fancy equipment that measures how much light a substance absorbs or transmits at certain wavelengths. The more purple the solution, the more light it absorbs and the less light it transmits. We know how much protein each standard has and the spectrophotometer will give each of these standards with an absorbance value. We don't how much protein each of our samples will have - but we will get an absorbance values for each sample. We can compare these to the standard curve our standards give and work out our protein concentrations. 

Some labs just call it a plate reader. 

28) Try to remember how to work the flipping thing.

29) Remember what absorbance* you want. 

*Absorbance refers to the wavelength of light the spectrophotometer is going to measure. You can in theory do transmission, but absorbance works better. The purple complex that was created absorbs light 562 nm so using this wavelength will give you accurate data. 

30) Let your spectrophotometer do its thing.  Almost forget to save the file it generates with the relevant absorbance values. 

31) Discard the plate. After all that work you don't need it anymore. 

32) Attempt to use these absorbance values to generate a standard curve*

*Some spectrophotometers do this automatically. It's a curve that plots the absorbance values for your standards against the protein concentrations. 

Looks a bit like this, Credit to Odinity 2018.

33) Breathe a sigh of relief when you see the R² number*.

*This number represents the 'coefficient of determination'. It basically says how well the data fits, giving you an idea of how reliable it is. You want to be about 0.990 - 1.000. An R² of 1.000 is perfect. 

34) Forgot all you know about maths and try to calculate how much protein you have in your samples*

  *How it works is based on the equation Y = Mx + C which is what you use to work out the gradient of a curve.  Y is the absorbance, X is protein concentration, C is your y-intercept and M is your gradient. You can use the standard curve to calculate the M and C. Those values are what you then use to calculate the X of the samples. But then you also need to multiple by the dilution factor -or how much you diluted the samples by in the first place. So, if you put 10 µl

 of sample with 90 µl of buffer, your final volume is 100 µl. Divide 100 by 10 and you get a dilution factor of 10. 

35) With a long sigh of annoyance, start planning the next stage - the gel electrophoresis. FUN.