Assay for Protein Quantification

Protein Quantification

Estimate the concentration of protein in a sample

Three types of protein quantification

Biuret Protein Assay

Lowry Protein Assay

Coomassie Blue (CB) Protein Assay

Which is the type of assay used in the PQ lab?

CB/Bradford Assay

Biuret Protein Assay

Alkaline copper solution assay

Lowry Protein Assay

Alkaline copper solution assay

CB/Bradford Assay

Dye-Binding protein assay

When the dye binds to protein, it changes color (from brown to blue) → intensity of the blue color = higher protein quantity

Qualitative test

Testing for the presence or absence of something, no numbers

Quantitative Test

Results are numerical

Is the CB assay qualitative of quantitative?

Quantitative → Uses the absorbance and a standard curveS to determine how much protein was absorbed

Spectrophotometer

Used to measure absorbance of light in a solution

How does a spectrophotometer work?

Shoots light through a solution

Detects how much of the light was absorbed

Higher light absorbance = more protein present

Reagents used in Bradford’s Assay

• Coomassie dye

• Protein extraction and dilution buffer

• Premade protein standards

• Protein sample fractions from the PF lab

What were the specific aims of the protein quantification lab?

• Learn protein assay principles

• Explore quantification methods

• Use Bradford (CB) assay to measure protein

• Study effects of lab reagents on assays​

Methods of the Assay for Protein Quantification lab

Protein standards and sample fractions were mixed with Bradford reagent, incubated in cuvettes, and their absorbance at 595 nm was measured using a spectrophotometer to determine protein concentration

What principles were outlined at the beginning of the protein assay lab?

Bradford’s Assay works by binding CB dye to proteins under acidic conditions which causes a color shift from brown to blue. More blue = More protein,