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

11
w can you study the expression of a gen

Transcript of How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western...

Page 1: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

How can you study the expression of a gene?

Page 2: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

Promoter activityRNA levelsProtein levels:

Western blotElisaEnzyme activty

Page 3: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

Proteins have complex structure

Page 4: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

Compare it to the structure of DNA:

Page 5: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

What is more labile?

Page 6: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

What factors affect the structure of your proteins?

What factors affect the activity of your protein?

Page 7: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

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)

Page 8: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

How do we measure enzyme activity?

Page 9: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

Enzyme activity assays:* Stop assay* Continuous assay

Page 10: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.

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

Could you have the opposite effect?

Page 11: How can you study the expression of a gene?. Promoter activity RNA levels Protein levels: Western blot Elisa Enzyme activty.