cardiovascular and haematology- year1 practical 1 Flashcards
how can you determine protein is pure?
enzymatic activity and protein concentration
what the concentration of protein tell you about absorbency?
the higher the protein concentration the higher the absorbency so less light is making it through
what needs to be kept the same when comparing samples?
the amount of protein used
after adding Bradford’s assay to protein what is the colour change?
red-brown to blue
what are the common proteins used for standard curves?
BSA and IgG- different amino acid compositions so different standard curve between the two
what are the conditions for the assay?
should be linear and apply to Beer’s Law
what is the definition of electrophoresis?
process of allowing movement of charged molecules through a solution by creation of an electrical field
how is the gel in electrophoresis made?
polymerisation of acrylamide
process of electrophoresis?
protein incubated with SDS forming ratio of SDS:protein. Protein migrates from cathode to anode
how are proteins denatured before electrophoresis?
SDS and thiol group added
disulfide bond broken
what is stacking gel?
sample condensed and put into bands
how is current carried in the gel?
current at the top carried by glycine, at ph 8.3 only 10% negatively charged.
At ph 6.8 no negative charge so current carried by Cl- forming concentration gradient of Cl-
how are proteins compressed into bands
Cl- below and glycine above them
what is the resolving and stacking gel?
resolving is the larger gel, stacking is the smaller
what is the Beer Lambert law?
measure of absorbance of chemical substance at a wavelength
