Lecture 2 Flashcards
In many cases of light microscopy, tissue samples must be chemically fixed and cut into thin slices (sections), why is this?
Chemical fixation stabilises and preserves biological samples for microscopy
Describe basic light microscopy and what it shows
- Descriptive differences between prokaryotes and eukaryotes, animal and plant cells etc.
- basic analysis of live or fixed cells
- magnifies cells up to 100X and resolves details to a resolutionof 0.2um.
- specimen must be prepared in a way that allows light to pass through
What does increasing the phase contrast allow us to see in light microscopy
flagella
What is differential interference contrast used for in light microscopy?
cells and organelles
Describe how fluorescent dyes work
- absorb light at 1 wavelength and emit it at another longer wavelength
- some dyes preferentially bind specific cellular components such as DNA
Two examples of fluorescent dyes that bind to specific components:
DAP1
Hoechst (Bisbenzimides)
What could dyes also be coupled to in order to use them to detect the distribution of specific proteins within fixed cells?
Antibodies
What are the advantages of confocal microscopy?
- V. precise
- higher revolutions
- optical sectioning can produce sharp images of 3D structure
What is confocal microscopy
uses fluorescence microscope but with laser light source
What is TIRF
Total internal reflection fluorescence microscopy
- can observe thin region of specimen in live cells
- especially good for selective visualisation of structures or events at/near plasma membrane in cells e.g. exo/endocytosis
Describe how green fluorescent protein works
-GFP can be added to a specific protein as a tag for fluorescence microscopy
-via recombinant DNA, protein transcribed fluoresce
-problems= can cause loss of function/misfolding and tags arent inert so you would have to prove that GFP hasnt altered protein
+Positives- works with live cells, is intrinsically fuorescent- can see GFP labelled structures move in real time
How does Electron Microscopy work?
Transmission EM: Thin sections stained with heavy metals for contrast:detailed subcellular structure
Scanning EM: good for 3D images
What is homegenisation?
Controlled rupture of plasma membrane
What is differential centrifugation?
=repeated centrifugation at progressively higher speeds will fractionate cell homogenates into their components
What gradient does velocity sedimentation use and for what?
- sucrose gradient
- for highly purified organelles such as ER and Mitochondria
What type of bonds may alter the mobility of a protein?
Disulphide bonds may alter mobility of a protein in a polyacrylamide gel
Describe SDS-PAGE
SDS Poly Acrylamide Gel Electrophoresis
for proteins
detergent SDS binds to and unfolds/denatures the proteins
-proteins are also treated with a reducing agent to cleave any disulphide bonds
-separates by size
Examples of reducing agents used in SDS PAGE
2-meracptoethanol
2ME
DTT
How is SDS PAGE visualised?
-when run on gel, you won’t be able to see anything, therefore shown in a stain
When is 2D gel electrophoresis used?
for V. complex samples with a lot of protein
separates on charge
Immunoblotting is used to….
Identify specific protein by using an antibody that recognises it
What can mass spectrometry be used for?
-identify one or more proteins in a gel slice by protein mapping
can be used for unknown protein
What do tryptic peptides do in mass spec?
provide a ‘unique’ fingerprint of a protein that can be used to identify it from a database by computational approaches
Drawbacks to SDS age?
- denaturing
- small scale
Describe chromatography
-separates native proteins on basis of differences in size,charge or substrate affinity (depending on column matrix)
Ion exchange chromatography separates by _______
charge
Gel-filtration chromatography separates by _______
size
In gel filtration column, larger proteins pass more _____ through column
quickly
What tag can be used to measure lateral mobility of membrane proteins?
GFP tags
How can antibodies be used as reagents
use antibody against protein A to immunoaffinity purify from a complex mixture in its native form
(dont really get what this means but it said it on a slide)
What coefficient can be calculated from a graph of rate of fluorescence recovery?
Diffusion coefficient
faster recovery indicates a larger diffusion coefficient
An antibody tagged with Gold is most suitable for……
electron microscopy
An antibody tagged with fluorophore is most suitable for……
fluorescence microscopy
An antibody tagged with enzyme is most suitable for……
immunoblotting
