Immunodepletion of Condensin from Xenopus Egg Extracts
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Transcript of Immunodepletion of Condensin from Xenopus Egg Extracts
IMMUNODEPLETION OF CONDENSINFROM XENOPUS EGG EXTRACTS
HSS Δcond. Δmock mockboiled beadsextractscond.
anti-cond.
anti-α-tub.
- 170
- 130
- 100
Smc2 Smc4
FLUORESCENCE MICROSCOPY: VISUALISING FITC-STAINED SAMPLES
Excitation filter (488nm+/-20 nm) Emission filter (525nm+/-25 nm)
Dichroic beam splitter(>495 nm)
Camera/Eyepiece
FLUORESCENCE MICROSCOPY:MICROSCOPE SET-UP
Emission filter
Excitation filter
Beam splitter
EXAMPLE: BRIGHT-FIELD MICROSCOPY OF A STAINED SAMPLE
Kidney ducts stained with hematoxylin (blue, basic extracellular matrix) and eosin (pink, acidic nuclei)
Source: MBC
BRIGHT-FIELD MICROSCOPYBased on differential absorption of light by objects
Absorption: Decrease in the amplitude of a light wave (i.e. object gets darker)
Absorption may be wavelength-independent or wavelength-specific (e.g. chloroplasts are green under the microscope, the amplitude of all other wavelengths is reduced)
Objects visible by bright-field microscopy are called “amplitude objects”
PHASE-CONTRAST MICROSCOPYThin objects (e.g. single cells) don’t absorb sufficient light to be good “amplitude objects”However, all objects shift the phase of a passing light-beam by a fraction of their wavelength. They are called “phase objects”.Using special optics this (invisible) phase shift can be converted into a (visible) amplitude shiftThis conversion is based in interference between the direct light beam and the phase-shifted light beam
EXAMPLE: PHASE CONTRAST
LIGHT PATH IN PHASE CONTRAST MICROSCOPY
Diffracted beamDirect beam
-1/4λ on the diffracted beam (passing through the specimen)
-1/4λ on the diffracted beam (passing through the retarder of the phase ring)
Net result: shift of 1/2λ of the diffracted beam results in negative interference between direct and diffracted beam apparent conversion of a “phase object” into an “amplitude object”
Δ1/4λ Δ1/4λ
TYPES OF LIGHT MICROSCOPY
Bright-field
Phase-contrast
Differential-interferencecontrast (DIC)
Source: MBC
Fluorochromes can be excited by a particular wavelength and emit light of a longer wavelength Stokes shift.
=heat
PRINCIPLE OF FLUORESCENCE
COMMON FLUOROCHROMES USED FOR BIOLOGICAL APPLICATIONS
PROBE DETECTIONAntibodies or nucleic acid probes can be conjugated to fluorescent dyes, such as FITC (fluorescein-isothiocyanate)
Fluorescent group
Reactive group for conjugation to other molecules via amine groups
SCALE BARS• all microscopic images must have a scale bar
• experimental determination of scale bar: take image of hemocytometer with squares of known dimensions (e.g. Thoma)
• calculate length from pixel as outlined below:
200 µm
CALCULATING MAGNIFICATION FOR DIGITAL MICROSCOPYPixel size: 6.8 µmCCD chip dimension: 1360 x 1024 pixelMicroscope magnification: 100x
6.8 µm/100 x1360=92.48 µm 6.8 µm/100 x 1024=69.63 µm
One image is 92.48 µm in length and 69.63 µm in height
Actin fibres in interphase cells
Stained with Phalloidin-FluoresceinDNA (DAPI stain) pseudocoloured in red
10 µm
Microtubules in interphase cell
Stained with anti-tubulin antibodies and secondary fluorescein antibodiesDNA (DAPI stain) pseudocoloured in red
10 µm
MACROPHAGE PHAGOCYTOSIS: SIGNALING THROUGH HETEROTRIMERIC G-PROTEINS
MACROPHAGE PHAGOCYTOSIS: CHEMOKINES ACT THROUGH HETEROTRIMERIC G-PROTEINS
Artificially activated by phorbol ester (mimics DAG)
19
Artificially elevated by ionomycin
DIACYLGLYCEROL AND PHORBOL MYRISTATE ACETATE
DAG
PMA
MACROPHAGE PHAGOCYTOSIS: E. COLI LIPOPOLYSACCHARIDE (LPS)
Lipid A
MACROPHAGE PHAGOCYTOSIS: LPS-INDUCED ACTIVATION CLUSTERS
LYMPHOCYTE PROLIFERATIONConcanavalin A:• Polyvalent lectin• α-D-mannosyl and α-D-
glycosyl binding• Mitogen• Polyclonal activation
(in contrast to antigen-mediated clonal expansion)
• Pleiotropic effects• Metabolic stimulation• Receptor clustering
(lipid raft)?
T-CELL ACTIVATION