0:00 the bread for test or bradford assay
0:04 is a technique used for protein
0:06 quantification
0:07 it is one among many colorimetric assays
0:11 used to determine the total protein
0:13 concentration
0:14 in a solution the bradford method is
0:17 based on a dye
0:19 named komasi brilliant blue in an acidic
0:22 solution
0:23 this reagent is protonated and has a
0:26 reddish or brownish color
0:29 in the presence of protein komasi
0:31 brilliant blue stably binds to them
0:34 the dye protein complex quickly takes a
0:36 blue color
0:38 required for this reaction is an acidic
0:41 environment the complex formation
0:44 results in a shifted absorption maximum
0:47 of the dye
0:49 the protein solution can be measured in
0:51 a spectrophotometer
0:53 at 595 nanometers
0:57 in the absence of protein there is no
0:59 change in color
1:01 the absorption maximum does not shift
1:04 how does komasi brilliant blue g250
1:08 interact with the proteins this
1:11 is kumasi brilliant blue in an acidic
1:14 environment
1:15 in the presence of protein the reagent
1:18 disrupts the protein structure
1:20 that exposes the amino acid side chains
1:24 the sulfonic acid groups of the dye
1:27 interact with the positive amino groups
1:29 of the protein
1:30 found in basic amino acid residues
1:33 present in lysine
1:34 arginine and histidine this results in a
1:38 stable form of the dye
1:40 and the color turns blue after dye
1:42 protein complex formation
1:44 but how do we determine the protein
1:46 concentration
1:48 the spectrophotometer measures the
1:50 optical density
1:51 at 595 nanometers
1:55 first the pure solution without any
1:57 protein content is measured
2:00 to calculate the protein concentration
2:02 of an unknown sample
2:04 we need a reference solution where the
2:06 protein concentration
2:08 is already known this is classified
2:11 as the standard in most cases the
2:14 protein
2:14 bsa is used for this different dilutions
2:18 of this standard are made
2:20 the optical density is measured for 1
2:23 microgram per microliter
2:25 and higher concentrations such as 2
2:28 and 4 microgram per microliter that
2:32 generates a linear curve
2:34 in which the optical density increases
2:36 proportionally
2:37 with protein concentration now
2:40 the sample with the unknown protein
2:43 content is measured
2:45 the spectrophotometer assigns the
2:47 optical density
2:49 and with extrapolation we can calculate
2:52 the protein concentration which will be
2:54 3 microgram
2:56 per microliter the bread for test has
2:59 advantages
3:00 and disadvantages one advantage
3:03 is that the komasi dye interacts quickly
3:06 with the proteins
3:08 the assay can determine protein
3:10 concentrations
3:11 in a very short time it is also
3:14 easy to use since it just requires the
3:17 bradford reagent
3:19 and a spectrophotometer the assay can be
3:22 performed at room temperature
3:24 in comparison to other protein
3:26 quantification techniques
3:28 the bradford method is extremely
3:30 sensitive
3:31 it can even detect protein
3:33 concentrations of
3:34 one microgram per milliliter one of the
3:38 disadvantages
3:39 is interference with detergents such as
3:42 sds since the commercial dye
3:46 reacts mainly with the basic amino acids
3:49 the assay
3:50 faces another limitation it is dependent
3:53 on the protein's amino acid composition
3:56 the bradford assay is just one among
3:59 many protein quantification techniques
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