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Quantifying Proteins Presentation

This document discusses methods for quantifying proteins. It begins by explaining that proteins must first be separated and purified from crude extracts before quantification. It then describes several common quantification methods, including Lowry assays using copper and Folin reagents to detect tyrosine and tryptophan, the Bradford method using Coomassie Blue dye, and electrophoresis to separate proteins by size and charge. Two-dimensional electrophoresis can further separate proteins by their isoelectric point and mass. The document concludes that various methods allow proteins to be quantified to determine their properties.

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25 views10 pages

Quantifying Proteins Presentation

This document discusses methods for quantifying proteins. It begins by explaining that proteins must first be separated and purified from crude extracts before quantification. It then describes several common quantification methods, including Lowry assays using copper and Folin reagents to detect tyrosine and tryptophan, the Bradford method using Coomassie Blue dye, and electrophoresis to separate proteins by size and charge. Two-dimensional electrophoresis can further separate proteins by their isoelectric point and mass. The document concludes that various methods allow proteins to be quantified to determine their properties.

Uploaded by

izziwhitley
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Quantifying Proteins

- What is protein quantification?


- Preparing proteins
- Methods for Quantifying Proteins
- Conclusion
What is protein quantification?
• Analysing proteins in a sample:

• Separating and purifying proteins

• Isolates one type of protein so it can be studied and differentiated


from other proteins

• Determine properties and interactions


Preparing proteins
• Proteins are separated by differing qualities:
• Size
• Charge
• Binding properties

• Obtaining proteins – crude extract

• Fractionation – based on different properties


Chromatography
• Proteins separated depending on their relative size, charge and
binding affinity

Ion-exchange Chromatography
• Exploits a difference in net charge of a protein
• Stationary phase is polymer beads with attached charged groups
• Proteins have a different affinity to stationary phase
• Proteins move down the column to form bands
Lowry assays
• Copper solution added
• Forms complexes with N in proteins (tryptophan and tyrosine)
• Folin-Ciocalteu reagent added
• Produces a blue-green colour from reduction
• Approximates amount of protein in a solution

• Disadvantages:
• Not compatible with many compounds
• Colour varies depending on the protein
• Colour isn’t proportional to concentration
Bradford Method
• Use Coomassie Blue indicator
• Added to protein solution
• Colour change from red to blue
• Absorbance measured – spectrophotometry
• Plot absorbance against a standard curve
• Beer-Lambert Law

• Not many compounds interfere with results


Electrophoresis
• Protein solutions pipetted into wells in polyacrylamide gel
• Migrate through it due to electrical potential across gel
• Separated by m/z and shape

• SDS – separated exclusively by mass


• Add dye that binds to proteins to show bands
• Approximate molecular weight by comparing sample to known
proteins
Two-dimensional Electrophoresis
• Isoelectric focusing – determining the pI of a protein

• Two-dimensional electrophoresis – combine electrophoresis and


isoelectric focusing
• Separates proteins with either the same molecular weight or pI
• Rotate gel after doing isoelectric focusing then carry out SDS
electrophoresis
Conclusion
• Proteins are quantified to determine their properties

• Before quantification they need to be separated and purified

• They can then be quantified by different methods, such as Lowry


assays, the Bradford method and Electrophoresis
Bibliography
Biology, P., Center, P., Library, P., Methods, P. and Assays, C., 2020. Chemistry Of Protein Assays | Thermo Fisher Scientific - UK. [online] Thermofisher.com. Available at:
<https://www.thermofisher.com/uk/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/
chemistry-protein-assays.html> [Accessed 28 October 2020].

Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical
Biochemistry, [online] 72(1-2), pp.248-254. Available at: <https://www.sciencedirect.com/science/article/pii/0003269776905273?via%3Dihub> [Accessed 28 November
2020].

Oliver H, O., 1951. Protein Measurement With The Folin Phenol Reagent. [online] pp.1-12. Available at: <https://www.jbc.org/content/193/1/265.full.pdf> [Accessed 28
October 2020].

Nelson, D. and Cox, M., 2017. Principles Of Biochemistry. 7th ed. Lehninger, pp.89-99.

En.wikipedia.org. 2020. Coomassie Brilliant Blue. [online] Available at: <https://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue> [Accessed 28 October 2020].

En.wikipedia.org. 2020. Bradford Protein Assay. [online] Available at:


<https://en.wikipedia.org/wiki/Bradford_protein_assay#Using_data_obtained_to_find_concentration_of_unknown> [Accessed 28 October 2020].

En.wikipedia.org. 2020. Quantitative Proteomics. [online] Available at: <https://en.wikipedia.org/wiki/Quantitative_proteomics#Applications> [Accessed 28 October 2020].

En.wikipedia.org. 2020. Lowry Protein Assay. [online] Available at: <https://en.wikipedia.org/wiki/Lowry_protein_assay> [Accessed 29 October 2020].

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