Research Flashcards
In vitro assays
used to measure the parameters of purified biomolecules such as protein
tightly controlled experiments performed outside a living organism
allows variables to be isolated, but that doesn’t accurately reflect behaviors of complex bio systems so must be confirmed by vivo
in vivo assays
conducted in whole living organisms with thousands of different molecules present
Western blot
technique used to detect presence of specific proteins
- load onto gel -> electrophoresis (moved by size)
- proteins transferred to protein-binding membrane to become immobilized
- any proteins not immobilized will become so with BSA or milk
- membrane incubated with antibodies that specifically bind protein of interest
- sometimes detected directly by markers or by addition of secondary antibody which binds to primary antibody - antibodies detected by fluorescence or chemoilluminescence
isoelectric point
pH when the amino acids has a net charge of 0
What proteins have the highest pI?
basic amino acids because the positive charge will need a higher pH to deprotonate the + side chains to become neutral
What proteins have the lowest pI?
acidic amino acids because the negative charge requires a lower pH for neutralization
What occurs during acetylation?
increases gene expression by freeing up histones
also neutralizes salts by neutralizing the amino acid’s positive charge
circular dichroism
an absorption spectroscopy method based on the differential absorption of left and right circularly polarized light. Optically active chiral molecules will preferentially absorb one direction of the circularly polarized light.
can assess secondary structure in proteins
gel elecrophoresis
separate based on size, charge, and shape
apply electric field with cathode and anode
larger molecules move slower so remain higher on gel
negative charge moves quicker toward anode than positive so will be further down gel
more aerodynamic shapes move quicker down gel
SDS-PAGE
type of electrophoresis that adds SDS which will denature the protein and cause them to be separated solely based on size
adds something that makes all proteins the same negative charge and will all equally want to move toward the anode, but will stop in different locations based solely on molecular weight
heavier objects remain at the top, lighter objects found at the bottom
reduced SDS-PAGE
type of electrophoresis that adds SDS and also a reducing agent so that disulfide bonds are broken
Native-PAGE
does not reduce or denature proteins- UNALTERED
applies electric field to original protein - maintains original structure
separates based on charge and mass
ex: double stranded DNA is not torn apart
Northern Blot
RNA
used after electrophoresis separates based on size
immobilized RNA with UV light
adds probe that is usually complementary strand with llabel to find specific RNA strand
Southern Blot
DNA
used after electrophoresis separates based on size
immobilizing DNA with UV light
separates into single strand
adds probe that will locate specific DNA using label
ELISA
enzyme-linked immunosorbent assay
used to see if proteins bind to specific antigens
direct ELISA
- antigen is immobilized
- primary antibody is washed over
- color change is reported by reporter gene if binding occurs
indirect ELISA
- antigen is immobilized
- primary antibody wash
- add secondary antibody that can recognize primary antibody
- reporting of color change by reporter gene
sandwich ELISA
see how much antigen is present
has primary and secondary antibody attach to the antigen
stronger color indicates a higher concentration of bound antibody reporters
precise
how reliable something is
if something is precise, you will have similar results obtained for repeated trials
the scale saying you weight 120 lbs every time you get on it
accurate
how correct the data represents real world value
this is assessed by comparison against gold standard
your phone saying it is 8:45am and comparing it to the world clock which says it is 8:45am
calibration must be
compared against gold standard
valid
both precise (very close in details) and accurate (giving correct information)
every time you get on the scale it says 120lbs. When you step on a scale that is calibrated corrected, it also says 120 lbs. So your scale is both precise and accurate so it is valid.
affinity chromatography
separates based on highly specific noncovalent binding interactions
only the target protein will be bound and then everything else will be washed away.
Second wash, the target protein will be washed into a solution that we can observe
cation exchange
columns are negatively charged and bind cations
so as the pH increases to become more basic (+) the more acidic peptides will elute sooner
anion exchange chromatography
columns are positively charged and bind anions
will bind negative atoms so positively charges peptides will elute faster
size exclusion chromatography
separates molecules by size
small molecules move slowly through size exclusion columns than do large molecules
OPPOSITE OF GEL ELECTROPHORESIS
Why do D-amino acids have a longer half life than L-amino acids?
proteases cannot act on peptides made of D-amino acids because of the odd angles the side chain protrudes
therefore enzymes essentially work exclusively with L-amino acids and no not affect D-peptides
stacking interactions
can stabilize secondary structures
occur between aromatic side chains - F, Y, P
proline in secondary structures
is rigid and can adopt cis-configuration which is conducive to tight turns in beta sheets
glycine in secondary structures
flexibility is favorable for beta turns
why do hydrophobic portions aggregate
disruption of protein’s native conformation may force exposure of hydrophobic residues to the aqueous environment. The hydrophobic residues are unable to form hydrogen bonds with water, so when they are exposed to aqueous environment, water molecules will form rigid network to satisfy hydrogen bond requirements and this is thermodynamically unfavorable
that is why hydrophobic molecules aggregate to decrease total surface area exposed to water
parallel beta sheets
parallel strands run in the same direction
N-terminal portion of one strand aligns with the N-terminal of the others
hydrogen bond pairs are slightly offset
lined by 360 degree loop
antiparallel beta sheets
individual strands run in opposite directions from each other so N-terminal of one strand lines up with C-terminal of neighboring strand
hydrogen bond pairs directly align
linked by 180 degree beta sheets
molecular chaperone proteins
help facilitate proper folding of other proteins
usually bind hydrophobic regions of nascent, misfolded, or aggregated proteins, preventing exposure of those regions to aqueous solvent
negative transcription factor
downregulates transcription
which amino acids are glucogenic?
leucine and lysine can be converted in to glucose through gluconeogenesis
glucogenic amino acid –> pyruvate –> oxaloacetate –> PEP
occurs through deamination
deamination
alpha amino acid + alpha ketoglutarate –>glutamate + keto acid
ex: alanine + alpha ketoglutarate –> pyruvate + glutamate
taking off the amine group and adding oxygen
irreversible inhibitors
react with nucleophilic side chains, forming covalent bonds to the enzyme
preincubation with an inhibitor
provides more time for covalent linkages to occur and can increase the level of inhibition because more enzymes will be inactivated
reversible inhibitors
bind target enzymes noncovalently
generally occur much faster, do not change with preincubation
nucleophilic amino acids
R-groups contain atoms which donate electrons when deprotonated
serine, threonine, cytoseine, tyrosine, and lysine
negative control
something is added where we know that no response will occur
ex: a study is looking at new types of fertilizer and how that will improve plant growth
negative control is adding no fertilizer. That way we can truly see if the treatment had an effect
other examples: placebo group, population that doesn’t receive any treatment
positive control
something added that we know will have an effect and we expect results to occur
ex: a study is looking at how new fertilizer improves plant growth
positive control is using a fertilizer that is known to improve growth by 20%. That way we can see if the treatment is effective
loading control
protein with high and ubiquitous expression
used to make sure that the protein has been loaded equally across all wells
should show up with all the wells having the same exact response
Co-Immunoprecipitation
common technique used to identify an interaction between two proteins
What type of SDS-PAGE is used for proteins?
proteins are smaller than DNA so loaded on a highly crosslinked polyacrylamide gel instead of agarose gel (which has larger pores)
