YouTube Transcript:
How to determine the protein concentration with the BCA Protein Assay

the bca protein assay also known as smith  assay named after its inventor is one of  
the most used techniques for the concentration  determination of protein briefly this method  
is a two component system belonging to the  colorimetric assays alongside the lori or  
bradford sa the characteristic of the copper-based  bca assay is a color change from green to violet  
in the presence of protein speaking about other  assays what are the advantages of the bca assay  
over the bradford test the benefit of the  bca protein assay is that it is compatible  
with detergents to a certain degree further the  bca assay is independent on the protein's amino  
acid composition but how does it work in the  first step a working solution composed of two  
different reagents is prepared reagent a includes  the carbonate buffer with a high ph of about 11  
that means that it is a highly alkaline  solution with a high concentration of  
o h minus ions it also contains the key molecule  by kinchoninic acid which the assay is named after  
reagent b is a cupric sulfate solution  those two reagents are mixed at a ratio of  
50 to 1 producing a greenish working solution the  second step requires the protein sample with the  
unknown concentration and a so-called protein  standard this standard is a serial dilution  
of a protein which has a very well-defined  concentration most standards use bsa as a protein  
this protein standard is utilized to create a  standard curve in the data analysis step in the  
next step both the protein sample and the bsa  standard is pipetted onto a micro tighter plate  
after adding the working solution with all  main ingredients for the colorimetric reaction  
the samples are incubated for 30 minutes at 37  degrees celsius meanwhile we can investigate which  
reactions inside those wells are happening in the  presence of protein the reactions of the bcasa  
can be divided into two steps in the first one  peptide bonds of the protein present in the sample  
reduce copper sulfate in an alkaline environment  to cupros in other words copper two plus cations  
are reduced to copper one plus cations the  higher the protein concentration the more  
copper one plus cations will be produced in this  reaction in the second step two bca molecules  
gelate with one copper plus ion this complex has  an absorption maximum at 562 nanometers which  
will be the reason why those reactions are visible  when we see the green turning into purple or blue  
after 30 minutes of incubation at  37 degrees the plate can be analyzed  
one can already see that this colorimetric  reaction is proportional to the protein  
concentration the standard without any protein  is still green whereas the highest concentration  
is dark purple to determine the protein content  in our sample of interest the plate needs to  
be inserted into a plate reader in which an  endpoint measurement determines the absorbance at  
562 nanometers the data of the bsa standard can  be plotted resulting in a standard curve the  
absorbance at this wavelength was also measured  for our protein sample of interest extrapolation  
is used to estimate the protein concentration of  this sample in practice a formula is generated  
after plotting the standard curve with that  formula and the absorbance at 562 nanometers  
the protein concentration can be easily calculated  if this video was helpful subscribe to the channel  
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