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