Protein Analysis. -Detection and quantification. Toby M Holmes

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1 Protein Analysis -Detection and quantification Toby M Holmes Clinical Research Unit UCD school of Medicine and Medical Sciences Mater Misericordiae University Hospital Dublin

2 Protein structure

3 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-immunosorbentassay Immunostaining Immunoprecipitation Immunoblotting (western blotting) Flow Cytometry/FACS analysis example of a 2D gel Proteomics - large scale screening of proteins in a cell, organism or biological fluid

4 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 Clinical cryostat for cutting tissue samples

5 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

6 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.

7 Antibody Structure IgG used as a model Also Ig A (dimeric), D, E and M (pentameric) Two chains light and heavy Variable regions (antigen binding sites) Fab Domain Variability of ~10 11 different antigens possible for more detail read Immunobiology by Janeway Constant Region (Effector Function)

8

9 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.

10 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 Monoclonal or Polyclonal monoclonals more specific and more expensive Cost- monoclonals can cost up to 400 each Does it require antigen retrieval can damage tissue morphology

11 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 VectorBlack

12 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

13 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 complete instructions.

14 Examples of ELISA Assays

15 Indirect ELISA

16 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

17 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

18 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 ( nm - semi-thins) Fixed or unfixed tissue/cells Methanol, acetone, 4% paraformaldehyde Clinical cryostat

19 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) Commonly used with polyclonal antibodies

20 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

21 Basic immunostaining method Sample preparation (rat fibroblasts) Blocking (3% Marvel or Serum of secondary ab host species) Primary antibody binds to antigen (monoclonal Ms a-rt actin) Secondary antibody detection (Gt a-ms FITC) Visualisation on a microscope (FITC filter)

22 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 antibody and DAB chromagen

23 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

24 Detection of fluorescently bound antibodies FITC - blue light TRITC - green light DAPI - purple light nuclear marker see arrow These are the filters that are commonly available.

25 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 3T3 cells stained with actin and dapi and transfected with gfp-tubulin.

26 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

27 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 Detection of specific protein using an antibody

28 Western blot

29 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 expressing different antigens

30 Proteomics The study of an organism's complete complement of proteins 35,000 genes in the human genome can code for at least ten times as many proteins This is due to post-translational modifications such as phosphorylation and ubiquitination Contact the UCD Proteome Research Centre at the Conway centre biology/proteomeresearchcentre/

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