BS2092 - Antibodies and Immunofluorescence Microscopy

Immunofluorescence microscopy

determination of intracellular localisation of a protein using antibodies

Antibodies

proteins produced by B lymphocytes as a defence against infection

Types of antibodies

• polyclonal

• monoclonal

Polyclonal antibodies

proteins that bind to antigens at multiple different antigenic determinants/sites as they contain a mixture of antibodies which recognise different antigenic determinants

Antigenic determinants/epitopes

short amino acid sequences found on the surface of folded proteins

Monoclonal antibodies

antibodies derived from a single clone of lymphocytes which produce only 1 type of antibody that binds to a single antigenic determinant/epitope

Features of polyclonal antibodies (PAb’s)

• inexpensive to produce

• skills required for production are low

• relatively quick to produce

• can generate non-specific antibodies

• recognise multiple epitopes on any one antigen

• can have batch-to-batch variability

Features of monoclonal antibodies (MAb’s)

• more expensive to produce

• training required to use technology

• hybridomas take a long time to produce

• generate large amounts of specific antibodies

• recognise one epitope on an antigen

• low batch-to-batch variability

• hybridoma made is constant and a renewable source of antibodies

Hybridoma

fusion of a single B lymphocyte and a cancer cell

Cross-reactivity of PAb’s

some of the epitopes that PAbs bind to may be present on other proteins

Batch-to-batch variability in PAbs

since PAbs are generated in an animal, once all the serum has been injected, a new animal is required to acquire more of that antibody leading to variability

Why are MAbs more favourable than PAbs for western blotting or immunofluorescence microscopy

Specificity

Use of PAbs/MAbs in immunofluoresnce

attachment of a fluorescent dye/molecule covalently bound to the antibody

Use of PAbs/MAbs in Western Blotting

attachment of a reporter molecule bound to the antibody

Fluorescence

a 3-stage process that occurs in fluorophores or fluorescent dyes involving the absorption of light then emission of light at a longer wavelength

Fluorescence process

• radiation of fluorophore with a specific wavelength causes molecule to be excited to a higher energy state

• molecule loses energy because of conformational changes and collision in the environment

• then returns to ground state, emitting a photon of light at a slightly longer wavelength

Typical time spent in excited state (for fluorophores)

1-10 ns

Relationship between emission wavelength and excitation wavelength

Emission wavelength is ALWAYS longer than Excitation wavelength

Why is the relationship between emission wavelength and excitation wavelength observed

the molecules have lost energy in the process of being excited and returning to the ground state

Fluorescent dyes used in Cell Biology

• small fluorescent dyes

• fluorescent proteins

• organelle-specific small fluorescent dyes

• cell signalling-specific fluorescent dyes

• pH-specific fluorescent dyes

Examples of small fluorescent dyes

• FITC (fluorescein isothiocyanate)

• TRITC (tetramethyl rhodamine isothiocyanate)

• Alexa Fluor dyes

Role of small fluorescent dyes

coupled to antibody molecules to detect localisation of a specific protein in a cell

Examples of fluorescent proteins

• GFP (green fluorescent protein)

• RFP (red fluorescent protein)

• YFP (yellow fluorescent protein)

Role of fluorescent proteins

used as markers or tags to study protein localisation in living cells

Difference between use of fluorescent proteins and small fluorescent dyes

fluorescent proteins can be used to look at behaviour of proteins in live cells while small fluorescent dyes can only be used to determine localisation in fixed/dead cells

Examples of organelle-specific small fluorescent dyes (and targets)

• Mitotracker — stains mitochondria

• Hoechst 33342/DAPI (4,6-diamidino-2-phenylindole) — stains DNA

Role of cell signalling-specific fluorescent dyes

used to monitor changes in the concentration and location of specific ions inside living cells

Examples of cell signalling-specific fluorescent dyes

• Fura-2

• Calcium Green

Role of pH-specific fluorescent dyes

measure intracellular pH in live cells

Fluorescence Microscope components

• light source

• first barrier filter

• beam-splitting mirror (dichroic mirror)

• second barrier filter

Fluorescence microscope light source

lamp filled with an inert gas (such as argon or xenon) to excite fluorophores

first barrier filter (in terms of FITC)

selectively only allows blue light with a wavelength between 450-490 nm to excite FITC

beam-splitting mirror (in terms of FITC)

• reflects light below 510 nm to focus it via objective lens onto FITC stained cells

• when light hits FITC molecules they emit fluorescent light

• fluorescent light emitted passes through the beam-splitting mirror which now allows light above 510 nm to pass through allowing light emitting FITC to pass through the second barrier filter

second barrier filter (in terms of FITC)

• barrier filter which only allows light between 520-560 nm, the emission wavelength for FITC

• allows us to view the light emitted by the molecules of FITC

• allowing us to view the green light emitted when we look through the eyepiece

Commonly used fixatives (for immunofluorescence microscopy)

• glutaraldehyde

• formaldehyde

Fixative mechanism

fixatives react with free -NH₂ groups of proteins, crosslinking them, thereby fixing adjacent protein molecules

Role of detergent

permeabilise the cell to create holes in the cell membrane to allow antibodies to enter the cell

Why must cells be permeablised

antibodies are too big (150 kDa) to penetrate the cell membrane

When do we use direct immunofluorescence microscopy

when we think the protein of interest is in high abundance

Direct immunofluorescence microscopy method

• cells are fixed (using glutaraldehyde or formaldehyde)

• cells are permeabilised by treating with a detergent

• incubate cells with an inert protein 1% w/v Bovine Serum Albumin (BSA)

• add FITC-conjugated antibody to target protein

• after incubation with antibody, wash cells with a buffer (phosphate-buffered saline) to remove any unbound antibodies

• observe cells under a fluorescence microscope

Purpose of 1% w/v Bovine Serum Albumin (BSA)

blocks non-specific protein binding sites to avoid getting a high background signal when Ab is added

Alternative fixation protocol

use of 100% pure alcohol (such as methanol) which fixes and permeabilises the cells simultaneously by dissolving the plasma membrane

When do we use indirect immunofluorescence microscopy

when we think the protein of interest is in low abundance

Indirect immunofluorescence microscopy method

• cells are fixed (using glutaraldehyde or formaldehyde)

• cells are permeabilised by treating with a detergent

• incubate cells with an inert protein 1% w/v Bovine Serum Albumin (BSA)

• add primary antibody (1° Ab) which will only bind to target protein

• add FITC-conjugated secondary antibody (2° Ab) that binds only to the primary antibody

• wash cells with a buffer (phosphate-buffered saline) to remove any unbound antibodies

• observe cells under a fluorescence microscope

Purpose of secondary antibody in indirect immunofluorescence

it amplifies the fluorescent signal to compensate for low abundance

Epitope tags

short peptide sequences we add to the protein of interest if there is no specific antibody to the target protein

Useful applications of epitope tags

• western blotting (immunoblotting)

• immunoprecipitation

• immunofluorescence microscopy

Common epitope tag examples

• Myc Tag (EQKLLISEEDL)

• HA tag (YPYDVPDYA)

• His6 Tag (HHHHHH)

Myc Tag precursor

human myc proto-oncogene

HA tag precursor

human influenza haemagglutinin (HA) glycoprotein

His6 Tag features

• can bind metal ions (such as Ni, Co, Cu, or Zn)

• useful for affinity chromatography

Purpose of using primary and secondary antibodies from different species

to ensure the secondary antibody detects the primary antibody as a foreign antigen

MCS

multi-cloning site containing a series of unique restriction enzyme sites used to cut the plasmid and gene to introduce the gene of interest into the cloning site, downstream of the sequence which codes for the epitope tag

Transient transfection

a rapid, temporary process of introducing exogenous DNA (e.g. plasmids) into cells

Transfection

a powerful analytical tool for studying gene function, gene regulation and protein function

Transient transfection process

• Mix purified recombinant plasmid DNA with a cationic lipid (in an Eppendorf tube)

• cationic lipid and the plasmid DNA will form a complex at room temperature

• once the complex is formed, add the transfection mixture to our culture dish containing cells

• leave the cells to take up the plasmid (takes about 16-24 hours)

• the cells need to undergo mitosis, when the nuclear envelope breaks down during mitosis, the plasmid will be incorporated into the nucleus and there it will be transcribed

• the mRNA exported from the nucleus will then be translated to produce an epitope tagged protein

• we can then detect the localisation of this epitope tagged protein by used a specific epitope antibody that recognises the epitope tag