P2 - Bradford Assay Flashcards
what is the max absorbance of bradford dye reagent?
595nm
what is the max absorbance of nucleic acid?
260nm
what is the A280 assay used for?
to roughly determine protein concentration, if it is not contaminated with DNA
when does the A280 absorbance trick not work?
when DNA is present in protein assay
how does biuret assay determine protein concentration?
detection of blue complex between biuret agent and peptide bond
which assay (bradford vs biuret) is better for smaller protein concentration detection?
bradford
which assay is better for detection of large protein concentrations?
biuret
how does bradford measure protein concentartion?
by measuring concentration of aromatic residues
how does biuret measure protein concentration?
by measuring amount of peptide bonds
If 45% of the light is transmitted by a sample in cuvette, the absorbance reading is?
0.35
If 60% of the light is transmitted by a sample in cuvette, the absorbance reading is
0.222
formula: 2-log(60)
in SDS page which comes first running or tracking gel?
running gel and stacking gel on top
what is running gel made from?
15% POLYacrylamide
what is stacking gel made from?
7% acrylamide
what is the function of acrylamide?
it is a mesh-like matrix to separate proteins based on size
what happens SDS when it is out of solution?
white crystals form
what is TEMED used for?
to catalyse polyacrylamide polymerisation (with APS)
what is butanol saturated water used for?
to allow gel to polymerise without matrix drying out
what is the need for sodium dodecyl sulfate? SDS
to eliminate charge on proteins
what is the need for Tris-HCl?
buffer - pH 8.7, keeps proteins linear
what is the need for acetic acid in SDS page?
provides an acidic environment - enhancing dye interaction
what is the need for acetic acid in SDS page?
provides an acidic environment - enhancing dye interaction
when is polyacrylamide considered a neurotoxin?
in solid and liquid form
why is kanamycin used?
prevention of bacterial contamination
