How to calculate Protein concentration using standard curve of BCA assay ? Easy steps

In this video, you will how to construct a Standard curve of BCA protein assay, and how to calculate protein concentration using a standard curve.
The Micro Bicinchoninic Acid (BCA) Protein Assay is a highly sensitive colorimetric assay that is compatible with detergent-solubilized protein solutions and is a modification of the Bicinchoninic Acid (BCA) Protein Assay for dilute protein samples (0.5-20µg/ml).
The Micro Bicinchoninic Acid (BCA) Protein Assay primarily relies on two reactions. Firstly, the peptide bonds in the protein sample reduce Cu2+ ions, in a temperature-dependent reaction, from the copper solution to Cu+
. The amount of Cu2+ reduced is proportional to the amount of protein present in the solution. Next, two molecules of bicinchoninic acid (BCA) chelate with each Cu+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm. The Micro BCA Assay uses
concentrated solutions and extended incubation times for the detection of dilute protein samples. The amount of protein present in a solution can be quantified by measuring the absorption spectra and comparing protein solutions with known concentrations.

For the BCA Assay procedure please watch this video:

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BCA Assay
Standard Curve construction
Calculation of protein concentration using a standard curve
How to construct standard curve in Skanlt Software
How to construct a Standard curve in Microsoft Excel

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[Mah-htsx]

Hi, thank you for this great and very detailed content. Would you please let us know the loading volume of your samples? How much µL are you loading in one well following your calculations?

[darkartsninja]

Thank you for the video! What do you do if you want to know the amount of sample for 20 ug of protein instead of 100 ug? Also, is the dilution factor 10 if you diluted 2 uL of sample into 92 uL of PBS?

[busragorgun]

There is an error in the calculation. Y is your abosrbance value and X is the unknown protein concentration. The opposite is given in the video.

[meleketzelalem]

hey, what did u use to calculate the unknown concentration of the sample? I’ve tried to figure out what it is by using either y = mx + c or the data using excel but I’m really stuck. Please help

[henrynnaemekaogbonna]

Hi can you explain the part where you calculated for the sample buffer. Is this a 4x or 2x sample buffer. If you were gonna calculate for 30ug for a 25ul. How do you put that in perspective with the 4x buffer

[beatrizbastosdemoraesthoma]

I think the blank BSA value should not be considered in the equation, right? The blank should be subtracted from the other measurements (all) and then the equation is made with these corrected values. Or not?