Colorimetric Assays and Standard Curves: The Bradford Assay Flashcards
What is a protein assay ?
Quantitation of protein in a sample
When would a protein assay be used ?
- During a purification procedure
- At the end of a purification procedure
- During enzyme kinetic studies
- During binding studies
- Comparative studies
What can a prtoein assay tell us ?
- Yield
- Fold purification
- Specific activity
What is the equation for specific activity ?
Activity of an enzyme/Protein concentration
What are the units of specific activity ?
Units per mg
If the enzyme is pure, what is the specific activity ?
Characteristic of that enzyme
What does low specific activity suggest ?
- Lots of contaminating protein
- Lots of inactive enzyme
What are some considerations when selecting a protein assay ?
- Is the assay fit for purpose
- Is the assay reliable
- Do you have the necessary instrumentation
- Is there a cost implication
- Is it fast
- Do reagents used in the purification interfere with the assay
- Has the method the required sensitivity
- Is the method destructive
What does protein absorb light maximally at ?
280nm
Why does protein absorb light maximally at 280nm ?
Due to the presence of tyrosine and tryptophan
What and why does phenylalanine absorb at ?
260nm due to aromatic ring
When can you use the beer lambert law to accurately determine the protein concentration ?
If you have a pure protein and you know the sequence of the protein
What is cysteine ?
The product of the cross linking of two cyesteine residues
What are colorimetric asssays based on ?
The use of a reference/standard protein
What considerations apply to colorimetric assays only ?
- Can you assume that the reference protein and your protein behave in identical ways ?
- Are you working within the linear range of the standard curve ?
- By its nature, the method is destructive, you will sacrifice protein, how much can you spare ?
What does the Bradford Assay involve ?
The addition of acidic dye, coomassie brilliant blue G-250 to the protein solutions
What does the coomassie dye bind to and what is the result ?
The dye binds to basic and aromatic amino acids resulting in a shift of the absorbance maximum from 465nm (brown) to 595nm (blue)
What is chromatography ?
A method for separating the components of a mixture on the basis of differences in physical of chemical characteristics
What type of chromatography for molecular mass ?
Gel filtration
What type of chromatography for charge ?
Ion exchange
What type of chromatogrpahy for specific bonding ?
Affinity chromatography
What types of molecules come out first of gel filtration chromatography ?
Large molecules
What is pI of protein ?
The point at which there is no net charge on the protein
What is a charge above pI ?
Positive
What is charge below pI ?
Negative
