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BSA

The document outlines the Bradford Assay, a method for determining protein concentration using Coomassie Brilliant Blue G-250 dye, which changes color based on its interaction with proteins. It details the absorbance shifts from 465 nm to 595 nm when the dye binds to proteins, and emphasizes the importance of measuring absorbance at specific wavelengths for accuracy. Bovine Serum Albumin (BSA) is highlighted as a standard protein used in these experiments.

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Dimple May Gianne Dumaguit
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36 views2 pages

BSA

The document outlines the Bradford Assay, a method for determining protein concentration using Coomassie Brilliant Blue G-250 dye, which changes color based on its interaction with proteins. It details the absorbance shifts from 465 nm to 595 nm when the dye binds to proteins, and emphasizes the importance of measuring absorbance at specific wavelengths for accuracy. Bovine Serum Albumin (BSA) is highlighted as a standard protein used in these experiments.

Uploaded by

Dimple May Gianne Dumaguit
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd

BSA

Determine protein content


20-200 microgram of protein
Maximum absorbance of an acidic solution of Coomassie brilliant blue G-250 shifts from 465nm
-595 nm WHEN BINDING TO PROTEIN OCCURS
Anionic form – stabilized by both hydrophobic and ionic interactions = VISIBLE COLOR
CHANGE IS OBSERVED

Coomassie G-250
- Cationic

Acidic Bradford reagent


(pinkish brown) doubly protonated / what it needs to bind stably to protein (protwin ubound)
465nm
(green) neutral
(blue) basic, anionic, unprotonated 595 nm

Reagent blank
- Contains everything except protein sample
- To “tare” /”auto zero”
The absorbance peaks of the protein-dye complex and the free red dye form are at 594 and 470 nm,
respectively. It is recommended to measure the absorbance at 590-600 nm, to achieve maximal
sensitivity to protein concentration change, and at 450-485 nm, to ensure linearization.

Unknown concentration is measured using Linear regression analysis y=mx+b


Y= measured absorbance
M= slope
X= unknown conc
B = y intercept

Standard preparations use C1V1 = C2V2


Bradford red amax= 470nm
Bradford w protein blue amax = 595
Green donated an electron but is not bound to the protein amax = 650nm

1st e – donated a charge groups of protein


Disrypts protein structure hydrophobic pockets exposure
Dye binds (sulfonic acid binds to amide)

Bovine Serum Albumin

 A serum albumin protein derived from cows


 Often used as a protein concentration standard in experiments
 The full length BSA precursor protein is 607 amino acids (AAs) in length, pH of1% solution is 5/2-
5.7
 Readily available

Coomassie Brilliant Blue G-250

 The different colors are a result of the different charged states of the acidic dye reagent

Principle of Bradford Assay

 Proteins containing basic amino acid (K,R,H) – ionic interaction – sulfate groupl and with
aromatic acids (F,Y,W) – pie stacking *hydrophobic inyeraction-non polar bind with
hydrophobic or Van der Waals interaction with Coomassie Brilliant Blue G-250 dye (binds
electrostatically in anionic form with basic AA side chains)
 There is an ionic interaction too between the positively basic amino acid side chains and the
negatively charged sulfonate groups on the dye molecule
 Protein-Dye complex causes a spectral shift from red-brown form of the dye (absorbance 465
nm) to the blue form of the dye (a595)

Bradford Method

- 20-220 microgram protein (required weight) = Boc=vine Serum Albumin (from cow)
- Intensity of color (absorbance) is directly proportional to concentration
-
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