BIOMG 4320 Prelim 1 | 2024
Resolution *** small dist b/t 2 pts can be distinguished;
D = .61λ/Nsinα, want lower D;
Light microscope 200nM
Bright field microscopy *** normal light type
Phase-contrast *** altered phase interference of unobstructed light; looks like cells are
surrounded by light
DIC *** 3D looking cells
Confocal *** 3D imaging with fluorescence, progessively capture full surface in focus
by removing out of focus; normal point-scanning vs. spinning disk
Deconvolution *** Takes normal fluor image, computer removes out of focus parts
PALM *** type of super res that only activates some GFP at a time, takes 1000s of
pics; STORM and FPALM
SIM *** superres that activate lines of GFP at multiple angles, deconstruct interference
STED *** supper res where depletion beam makes stim beam even smaller
TIRF *** fluor microscopy for resitrcted focal plane near coverslip
2-photon excitation *** image deep tissue like brain of living mouse
light sheet microscopy *** rapidly image large volume e.g. living itssue
IgG digests *** papain: 2xF(ab) monovalent; pepsin: F(ab)₂ bivalent
B cell gen *** hematopietic SC's: assym div one SC on lympoid progenitor =>
intermediate progenitors => ; regulated by cytokines
, clonal expansion *** activated when antigen binds receptor on B cell, if antigen has
multiple epitopes, multiple expansions=> polyclonal
Linear epitopes *** better for denaturing gels like SDS-PAGE
generating hybridomas *** auzotrophic myelomas fused with B-cells from X immunized
mouse, only fused cells survive on -purine media, are monoclonal
EM *** up E, down λ, better res .1nm, TEM and SEM; need to dehydrate, fix, stain in
vacuum
negative stain TEM *** heavy metal stain, outlines small particles, some internal struct
if penetrates (proteins)
heavy metal shadowing TEM *** smaple absorbed on mica, coat with platinum, then C,
dissolve mica and sample, view topology
cryo TEM *** frozen samples, no stain, fixing, dehydration needed. comp avgs 100s of
pics, can also tilt axis for better 3D imaging
immunoEM *** dots where antigen is using ab whose Fc regio attached to gold beads
with ProteinA attached
FACS *** cells sorted by level of fluoresce (flow cyt basically)
subcellelar fractional differential centrifugation *** sequentially purify by removing pellet
at higher speeds (g force) and increased time
equilibrium centrifugation *** sucrose density gradient
IP *** use Protein A coated agarose beads to capture Fc
Mass spec *** m/z ratio; compare mass of each ion peptide to all predicted; MS/MS
psectrum of further ionizations of major peak fragments from first round
primary mammalian cell culture *** works for non-terminally differentiated, cells directly
isolated from tissue, have a finite number of divisions; not practical for obtaining large
amounts of protein; passage by transferring to new plate every 3 days (subculturing)
oncogenic transformation *** esp in rodent primary cultures, after period of senescene,
immortal cell lines with emerge due to oncognesis
Resolution *** small dist b/t 2 pts can be distinguished;
D = .61λ/Nsinα, want lower D;
Light microscope 200nM
Bright field microscopy *** normal light type
Phase-contrast *** altered phase interference of unobstructed light; looks like cells are
surrounded by light
DIC *** 3D looking cells
Confocal *** 3D imaging with fluorescence, progessively capture full surface in focus
by removing out of focus; normal point-scanning vs. spinning disk
Deconvolution *** Takes normal fluor image, computer removes out of focus parts
PALM *** type of super res that only activates some GFP at a time, takes 1000s of
pics; STORM and FPALM
SIM *** superres that activate lines of GFP at multiple angles, deconstruct interference
STED *** supper res where depletion beam makes stim beam even smaller
TIRF *** fluor microscopy for resitrcted focal plane near coverslip
2-photon excitation *** image deep tissue like brain of living mouse
light sheet microscopy *** rapidly image large volume e.g. living itssue
IgG digests *** papain: 2xF(ab) monovalent; pepsin: F(ab)₂ bivalent
B cell gen *** hematopietic SC's: assym div one SC on lympoid progenitor =>
intermediate progenitors => ; regulated by cytokines
, clonal expansion *** activated when antigen binds receptor on B cell, if antigen has
multiple epitopes, multiple expansions=> polyclonal
Linear epitopes *** better for denaturing gels like SDS-PAGE
generating hybridomas *** auzotrophic myelomas fused with B-cells from X immunized
mouse, only fused cells survive on -purine media, are monoclonal
EM *** up E, down λ, better res .1nm, TEM and SEM; need to dehydrate, fix, stain in
vacuum
negative stain TEM *** heavy metal stain, outlines small particles, some internal struct
if penetrates (proteins)
heavy metal shadowing TEM *** smaple absorbed on mica, coat with platinum, then C,
dissolve mica and sample, view topology
cryo TEM *** frozen samples, no stain, fixing, dehydration needed. comp avgs 100s of
pics, can also tilt axis for better 3D imaging
immunoEM *** dots where antigen is using ab whose Fc regio attached to gold beads
with ProteinA attached
FACS *** cells sorted by level of fluoresce (flow cyt basically)
subcellelar fractional differential centrifugation *** sequentially purify by removing pellet
at higher speeds (g force) and increased time
equilibrium centrifugation *** sucrose density gradient
IP *** use Protein A coated agarose beads to capture Fc
Mass spec *** m/z ratio; compare mass of each ion peptide to all predicted; MS/MS
psectrum of further ionizations of major peak fragments from first round
primary mammalian cell culture *** works for non-terminally differentiated, cells directly
isolated from tissue, have a finite number of divisions; not practical for obtaining large
amounts of protein; passage by transferring to new plate every 3 days (subculturing)
oncogenic transformation *** esp in rodent primary cultures, after period of senescene,
immortal cell lines with emerge due to oncognesis
