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
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