The Genetic Code, Proteins, and Techniques to Study Proteins Flashcards
How did we determine that the mRNA was read in triplets?
When there was a single nucleotide insertion, gene function was disrupted. Same with having 2 nucleotide insertions. But with 3 nucleotide insertions the sequence was ultimately read as the wild type sequence downstream of the mutations
How did we determine which codons encode which amino acids?
Created synthetic mRNA with a continuously repeating sequence and added it to a bacterial extract that contained ribosomes and amino acids. They then examined the polypeptide created, and repeated the experiment until all 64 possible combinations of triplets had been sequenced
There are 64 possible triplet codon combinations, but only 30-61 types of tRNA in cells. How can there be an anticodon to bind to all possible codons?
The 5’ end of the anticodon/the third base of the codon is promiscuous. It doesn’t need to be complementary as long as the first two bases are, which might contribute to the degeneracy of the genetic code
What are the two regions on an antibody?
The constant region is exactly the same for every antibody from that organism, and the variable region is the part that binds to the antigen and is very specific
What is the difference between a primary and secondary antibody?
A primary antibody will bind to the protein of interest, and a secondary antibody binds to the primary antibody and is labelled in some way
What sort of sample preparation is required for an SDS-PAGE gel?
The proteins need to be boiled to denature them, have a reducing agent added to break disulfide bonds, and add SDS to coat the proteins with an even negative charge
Why would we do staining with Coomassie instead of doing a Western blot?
To look for differences in expression of highly abundant proteins
How does a Western blot work?
All proteins are run out on a gel, transferred to a membrane, have the primary antibody added, then the secondary antibody that is visualized
What does a Western blot tell us?
If a protein is being expressed, it’s size, and is quantitative
How does immunofluorescence work?
Primary and secondary antibodies are added to cells and visualized
What does immunofluorescence tell us?
Where in cells a protein is being expressed, but it isn’t quantitative
