BCA Protein Quantitation Assay (Using Thermo Fisher #23227)

Utility: This is a quick assay performed to quantify the concentration of total protein in samples. This assay is traditionally used to measure protein concentrations in cell lysates so they can be normalized to each other for western blotting. This is useful because in western blotting you want to load the same protein content from each sample so it's simpler to measure relative expression of your protein of interest. 

How it Works: Peptide bonds in proteins of your samples reduce Cu2+ ions from the copper(II) sulfate reagent to Cu1+, then two molecules of bicinchoninic acid form a complex with each Cu1+ ion which yields a purple complex that absorbs light at 562 nm. The assay is measured by passing this light through the samples and measuring how much was absorbed. 

Watch Out For: This assay functions via a reduction mechanism, so inclusion of any reagents in your sample such as DTT or BME would interfere with the reaction. It's best to add these after performing this assay, when preparing your samples for SDS-PAGE. If these reagents are present they can be removed via Zeba desalting columns. 

Summary: This protocol is re-worded from that of the manufacturer (Thermo Fisher) and includes an updated standard curve preparation scheme. This was done since the initial protocol yielded different final volumes for each standard, which was problematic because this assay uses the same volume of every standard. With this updated dilution series you won't end up with 1 standard that is used up while having 7 others which still have residual volume. 

Quick Pro Tips: 

• You can prepare your own stock solution by diluting BSA in PBS in order to save reagent. This protocol is to be used with Thermo Fisher #23227 which comes with its own BSA standards, but more can certainly be prepared through simple weighing and dilution.
• This assay can be performed in uncoated 96-well plates, they don't have to have the tissue culture coating for growing cells. This can significantly save on resources.
• Samples can be diluted 2x to use half as much volume. This can be useful when analyzing a sample you don't have a large amount of.
• If you're unsure of the concentration of your samples and you're worried about overshooting the curve, it's wise to prepare 2x, 4x, and 8x dilutions of your samples. This will drastically increase the likelihood that you'll get a measurement that's within the curve range.
• When putting your plate into a plate reader, it will also fit when rotated 180 degrees. This means you can use the same plate for 2 assays and in one fell swoop halve your plate usage. This is of course only possible if there are ample empty wells after the first usage of the plate.

Photo: This is what the assay looks like when it's finished. Standard curve is on the right, and the gradient from dark purple to clear shows you a dilution series was made. 

Standard Curve Preparation:

Diluent should be the same liquid that your samples are comprised of. This is usually your lysis buffer + protease or phosphatase inhibitors which you used. These standards should be prepared in 1.5 mL tubes and stored -20C before usage. This series is starting from a 2,000 ug/mL stock, which comes standard in this kit. 

BCA Assay

1. Thaw BCA standards on ice 2-3h beforehand, or if you're in a rush, place them on the sash of a fume hood and check their thawing status every 2-3 minutes. We want them to stay cold.
2. Aliquot 10 uL of standards and samples in duplicate into flat bottom clear 96 well plate. Half volumes of either can be used to save on reagent. I've found using 5 uL of each works just fine.
3. Turn on microplate reader, and open analysis software pre-programmed with well positions and concentrations of standards as well as samples.
4. Prepare BCA reagents by mixing 1 part Reagent B with 49 parts Reagent A and vortexing, utilizing sufficient volume for 80 uL per well of sample and standards with 20% excess. For example, if I had 20 samples, I would prepare 1,920 uL of BCA reagent, comprised of ~1882 uL of Reagent A and 38.4 uL of Reagent B.
5. Using multichannel pipette quickly add 80 uL of mixed BCA reagent to all wells containing sample, jiggle plate gently on countertop, and put plate in 37C incubator for 28 min.
6. Take plate out of incubator and shelter from light and allow to reach room temp for 2 min.
7. Place plate into plate reader and quantitate protein by measuring absorption at 560 nm
8. Use values obtained to normalize samples for western blotting, using lysis buffer to bring up volumes to equivalence.

Assay Photos and Additional Tools

Most plate readers will have an accompanying software which will construct a standard curve for you. I've included one below for reference: 

And these are the standard curve fitting details. 

This one had a particularly good R^2 of 0.997. R^2 is a measure of how well correlated your standard curve is. An R^2 of greater than 0.95 is acceptable. If not, it's recommended to repeat the assay, and if this happens again, re-prepare a new set of standards. 

The plate reader will use the standard curve to calculate the concentrations of your samples. It's important here to include any dilution factors your samples may have, and scale them up by that amount. Also, make sure you're using concentration values, not absorbance values. 

Once you have your concentrations from the BCA assay, proceed to preparing your samples for running on a gel. Use this calculator I've made to make your sample process much easier: https://www.researchhub.com/post/2034/gel-loading-calculator-for-western-blotting