Studying protein expression Flashcards
What tools can be used to find an expressed gene and its components?
Search databases and literature
Map transcriptome
Measure transcript under different conditions (RT-PCR) as well as the enzyme activity
What tools can be used to study regulation of a protein?
Change the 5’ region and fuse with reporter (GFP/lux) > analyze changes of transcript on gel and/or cells
DNA affinity assay to figure out the relevant DNA binding proteins
Electromobility shift assay (EMSA): Determine that the DNA and protein actually form complexes, based on migration through gel
Analyze if actual regulation occurs – grow wt, knock-out and overexpression to determine!
What tools can be used to see if there is a connection between regulation of a certain protein expression and something else?
Look at a broader scale if something else is co-expressed with the regulating gene product – investigate transcripts with qPCR, grow side-by-side to see differences and do a total 2D gel and look for changes in composition or amounts!
2D gels: Total protein to see differences between conditions, purified protein to see different PTMs
