Clinical Research Unit
UCD school of Medicine and Medical Sciences Mater Misericordiae University Hospital
Dublin
Protein Analysis
Protein Analysis
-Detection and quantification
-Detection and quantification
General methods
• Protein precipitation assays
– Bradford and Lowry assays for total protein content
• Electrophoresis - separation of proteins – 1D & 2D gels, HPLC- high pressure liquid
chromatography
• Antibody based analysis
– ELISA - enzyme-linked-immunosorbent-assay
– Immunostaining
– Immunoprecipitation
– Immunoblotting (western blotting) – Flow Cytometry/FACS analysis
• Proteomics - large scale screening of proteins in a cell, organism or biological fluid
Catherine McAuley facilities
• Good cell culture and
molecular biology setup
• Cold room and ultra low
temp freezers
• Complete UV-Visible light spectrophotometer
and 96 well plate washer
• Gel electrophoresis kits
• Inverted and fluorescent microscopes
• Digital cameras for the above
Antibodies
• Proteins with extremely specific binding
properties to other proteins
• Extremely useful in detecting their target proteins
• Can be used to quantify their target proteins
What are Antibodies
• Glycoproteins (Immunoglobulins, Ig) naturally produced in response to invading foreign particles (Antigens).
• Critical role in immune defence against infection and disease. • Each antigen may have many epitopes (binding sites)
• Each antibody binds to a single epitope on an antigen (very specific)
• Antibodies are comprised of antigen and cell binding regions • Monoclonal or polyclonal?
– monoclonal ab are produced artificially from a single cloned plasma cell and are specific for a single epitope.
– polyclonal ab are produced by many plasma cells as occurs naturally and bind many epitopes of an antigen.
Antibody Structure
• IgG used as a model
• Also Ig A (dimeric), D, E and M (pentameric)
• Two chains light and heavy
• Variability of ~1011
different antigens possible
• for more detail read Immunobiology by Janeway
Variable regions (antigen binding sites)
Constant Region (Effector Function)
Fab Domain
Immunochemical techniques
• Each technique presents the antigen in a different physical context and therefore requires different properties from the corresponding antibodies.
• Immunostaining - antigen is immobilised in tissue, its native but complex cellular context.
• Immunoprecipitations - antigens in solution surrounded by a huge number of contaminants.
• Immunoblotting - antigens left denatured and partially purified bound to a solid support such as agarose or magnetic beads.
• An antibody used for ELISA may not be suitable
for immunostaining.
Choosing the correct 1°Ab
• Read the literature. Has it been done before, will it work on similar targets.
• Target
– Species Hmn, rt, ms, rbbt, pg
– Tissue, secreted growth factor or growth factor receptor, intracellular or extracellular proteins.
• Processing
– live cells, fixed cells, cryosections, wax or resin embedded
• Specificity
• Commercial or gift (not everything is on the market)
custom antibodies can be created for around €3-4000
• Monoclonal or Polyclonal
– monoclonals more specific and more expensive – Cost- monoclonals can cost up to €400 each
• Does it require antigen retrieval
Choosing the correct 2°Ab
• Specific for 1°Ab species
• Will not cross react with tissue/cells
• Is enzyme amplification required
– Avidin-biotin complex
• How are we going to detect it
– light, fluorescent or confocal microscopy
• Fluorochromes - fluorescent tags
– FITC - blue light
– Texas Red/ TRITC - green light
– DAPI - Darker blue light (Nuclear stains)
• Chromagens - chemical tags – Vector ABC kits
– Diaminobenzidine (DAB) - brown – NovaRed
ELISA
• Used to quantify solubilised or secreted proteins
• Enzyme-linked-immunosorbent-assay
• Not Elisa or Eliza
• Used to detect antigens in a sample either
quantitative or qualitatively
• Generally uses a 1º ab to detect the antigen and
a 2º enzyme linked ab to amplify the signal
allowing very low levels of protein to be detected.
• Buy the kit
Four types of ELISA
• Indirect - 1º ab ⇒2º ab. • Direct - 1º ab. labelled. • Sandwich - 2x 1º ab
sandwiching antigen ⇒2º ab.
• Competition (or inhibition) detects and monitors
antigen concentration using competitive binding of ab to free or immobilised antigen. • Each kit will come with
Examples
of ELISA
Assays
Multiplex antibody arrays
•MSD – Meso Scale Discovery
multi-spot sandwich immunoassay
•Measure up to nine antigens in a single 96 well plate
•Capture antibody is precoated on specific spots of a MSD multi-spot plate
• Antigen levels are quantified using a specific detection antibody labelled with electrochemoluminecent sulfo-tag which emits light under electrical stimulation • Equipment available at the Conway institute
Immunostaining
• Used to detect proteins bound to cell membranes,
structural proteins, extracellular proteins in
tissues, cytokine receptors etc
• Not good for detecting secreted proteins in tissue
as they may appear as a smear
• Detection of secreted protein receptors may be
useful
Immunostaining
• Staining of Cells in culture
– 8 well culture slides or flasks
• Staining of tissue sections
– Cryosections - frozen and cut using a cryostat (5-20µm) – Paraffin embedded- cut on a
microtome (2-20µm)
– Resin embedded - cut on an ultramicrotome (50500 nm -semi-thins)
• Fixed or unfixed tissue/cells
– Methanol, acetone, 4% paraformaldehyde
Blocking
• Reduce background staining of non-specific proteins. • Not always required (check protocol).
• Generic blocking agents e.g. 3% Marvel or bovine serum albumin (BSA)
• Specific blocking agents e.g. gt serum (usually the
serum of the host animal to the secondary antibody is used)
Visualisation
• Fluorescent linked secondary
• Enzyme linked secondary
– Horse radish peroxidase (HRP) - normal light – Alkaline Phosphatase (used in many ELISA kits) – Glucose oxidase
• Counterstaining
– Hematoxylin – Methyl Green – Fast Red
– Nuclear marker such as DAPI
Tumour cells stained with specific cytoplasmic ab and hematoxylin QS counterstain
Basic immunostaining method
Visualisation on a microscope (FITC filter) Secondary antibody detection (Gt a-ms FITC) Primary antibody binds to antigen(monoclonal Ms a-rt actin) Blocking
(3% Marvel or Serum of secondary ab host species)
Sample preparation (rat fibroblasts)
Transmitted light microscopy
• Uses either a standard upright binocular
microscope or an
inverted microscope. • Used with basic
histological stains of
tissue or phase contrast visualisation of live cells. • Cell chamber slides allow
easy viewing of cell morphology.
• Correct setup of light path important.
Pictures of both microscopes
Germinal centers in a human lymph node treated with monoclonal anti-CD21
Fluorescent microscopy
• Uses filters to visualise specific wavelengths and stimulate fluorescently tagged antibodies
• Fluorescence fades as it is viewed so use anti-fading mounting media
• Requires a low light camera for imaging
• Camera controlled using Image pro Plus™ image analysis program
Detection of fluorescently bound
antibodies
• FITC - blue light
• TRITC - green light
• DAPI - purple light
These are the filters that are commonly available.
nuclear marker see arrow
Multiple staining
• Take multiple pictures using different filters.
• Use photoshop to combine colour channels.
• Used to show co-localisation of proteins and/or protein receptors.
• Image analysis programs can help automate this process • Confocal microscopy excels at
this (Conway inst.).
– Contact Anne Cullen – [email protected]
3T3 cells stained with actin and dapi and transfected with gfp-tubulin.
Immunoprecipitation
• Used to isolate and purify an antigen from cells or tissues using an ab attached to a sedimentable matrix • Four stages – Immobilisation of ab – Solubilisation of ab – Immunoprecipitation – Analysis of precipitated protein
Western Blotting
• Used to detect and identify proteins in samples
like cell pellets, cell culture supernatants and
tissue homogenates.
• Four basic steps
– Preparation of sample (denature proteins)
– Separation of proteins by size (electrophoresis) – Transfer of proteins onto solid membrane
Flow Cytometry/FACS
• High throughput automated quantification of cells in
solution either using fluorescence or light scattering
• Fluorescence activated cell sorter (FACS)
• Situated at the Conway Institute, UCD Belfield
– Dr Alfonso Blanco
• Used to quantify cell surface markers
• Sorts and counts cells
