MMBIO 241 Flashcards
How does electrophoresis work?
the gel box has an electric field with a positive and a negative end. DNA is negatively charged so it migrates to the positive end
What are some real world applications for DNA fingerprinting?
- Crime scene investigation
- Paternal determination
- Identifying different species
- Determining ancestry
- Creating evolutionary trees
What does the primary antibody do in ELISA?
bind to antigen
What does the secondary antibody do in ELISA?
bind to primary antibody and has the enzyme HRP attached
What does the HRP enzyme do in ELISA?
converts substrate into blue color
What does the substrate do in ELISA?
turns blue by enzyme
What does the antigen do in ELISA?
attaches to the sides of the well
What are some real world application for ELISA?
- Home pregnancy tests
- Disease detection
- Drug detection
What is the role of SDS?
denatures proteins and lyses cells to get to the DNA
What is the role of Protease K?
attacks proteins and lyses cells to get to the DNA
What is the role of EDTA?
chelates to metal cations which inhibits DNAses so they cannot destroy our DNA
Why is coomassie brilliant blue dye used in conjunction with the spectrophotometer to determine the protein concentration of the sample?
the dye interacts with the protein causing an absorbance shift from 465 nm to 595 nm, this shift can be measured, not much is read at 595 nm
Why do you need to use a standard curve to determine protein concentration by spectrophotometry, but not for DNA conc.?
Because the makeup of DNA is uniform, a constant was created for A260 (50 ug/ml) and a standard curve is not necessary.
Proteins do not have a uniform makeup, so it is necessary to use protein standards together with some means of measuring the amount of protein like coomassie blue
How does the SDS page work? Why do proteins migrate to the positive electrode?
SDS first denatures the protein and then gives it a uniform negative charge which is why it moves to the positive electrode
Why would a larger protein migrate more slowly than a smaller protein?
It is harder for something bigger to move through the pores of the gel
