Studying protein expression Flashcards
(3 cards)
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
