How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

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Presentation transcript:

How can you study the expression of a gene?

Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty

Proteins have complex structure

Compare it to the structure of DNA:

What is more labile?

What factors affect the structure of your proteins? What factors affect the activity of your protein?

How do we measure total amount of proteins? The Bradford assay, a colorimetric protein assay, is based on an Assay absorbance shift in the dye Coomassie when bound to arginine and hydrophobic amino acid residues present in protein. The anionic (bound) form of the dye is blue and has an absorption spectrum maximum at 595 nm. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. (Wikipedia)

How do we measure enzyme activity?

Enzyme activity assays: * Stop assay * Continuous assay

Could you have differences in enzyme activity of a particular protein but its RNA levels remain constant? Could you have the opposite effect?