Practical molecular biology: PROTEINS

Dr. Alexei Gratchev

PD Dr. Julia Kzhyshkowska

Literature

Current protocols in molecular biology www.methods.info www.dako.com

Protein analysis Immunohistochemistry (IHC)

Principles of protein detection Qualitative and quantitative analysis IHC and IF FACS

Protein Problem Molecular Weight (MW); how many forms; charge and shape;

Posttranslational modifications;

Biosynthetic pathways, half-Life, degradation pathways;

Intracellular localisation; trafficking pathways

Integration in protein-protein networks; interaction with DNA, RNA, lipids;

Expression profile in cells and tissues;

Biological function: one or many, regulated or constitutive, intracellular or extracellular, ubiquitous or cell-type specific;

Proteins in pathology: biomarkers and molecular mechanism of a disease;

Therapeutic protein targeting

Alzheimer disease and plaques

Alzheimer disease (Morbus Alzheimer, MA) is neurodegenerative disease, affecting individuals older than 65 years . MA is responsible for appr. 60% (24 millions worldwide ) of dementia.

Characteristic symptoms are progressive degradation of cognitive capacity, reduction of daily activities, behavioural changes.Plaques are formed in the brain of a patient several years before first clinical signs of neurophysiologic changes can be detected,These plaques are formed by wrongly folded beta-amyloid-(Aβ-)peptides

1. Amyloide-precursor protein (APP) is constitutively exposed on the surface of a neuron. Cleavage of APP is performed by -secretase results in the production of soluble APP-s

2. Internalisation of APP results in the pathological intracellular APP processingand secretion of APP-s

3. Accumulation of APP-s results in the formation of plaque in the brain

Protein detection

Direct sequencing (for purified protein);

MW: motility in the gel (usually for denatured proteins) or gel-filtration chromatography (usually for native proteins) (for purified protein or protein complex with limited amount of components);

Immunological detection (for purified proteins, protein complexes and crude material like cell lysates or tissue extracts)

Enzymatic activity

Protein quantification Total protein amount in the sample (spectrophotometric detection);

ELISA: measurement of concentration of specific protein

Enzymatic reaction: measurement of activity, does not necessarily correspond to the concentration of an enzyme

FACS: measurement of positive cells however relative quantification of protein amount in the cell

Semi-quantitative and relative methods: Western blotting, IHC, IF

Immunological detection of the protein

Methods: Immunohistochemistry (IHC), Immunofluorescence (IF), Enzyime-Linked Immunosorbent Assay (ELISA) Western Blotting (WB), Immunoprecipitation (IP), Fluorescence Activated Cell Sorting (FACS)

Principle of recognition

primary antibody binds to specific epitope (one or several) in the protein

Principle of detection

primary antibody or secondary antibody that recognise primary antibody is labelled (examples: HRP for IHC and Western blotting, fluorescent dye for IF and FACS)

Material for IHC and IFFresh or frozen Tissue sections; Cells grown on cover slips; Cells sedimented on object glass

using cytospin centrifuge

Paraffin embedded Tissue sections

Advantages Disadvantages

Limited time of storage

Retrospective analysis is not possible

Advantages Disadvantages

Antigen-retrieval has to be designed individually for most of antigens

Only limited amount of labeled primary antibodies recognize retrieved antigen

Antigens are in a good shape, and most of primary antibodies can be used

Intracellular localization studies are possible even in tissue sections

Extremely long storage time,

Retrospective analysis can be done on archive material

Methanol-Acetone FixationFix in cooled methanol, 10 minutes at –20 °C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at –20 °C. OrParaformaldehyde-Triton FixationFix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 2-10 minutes. OrParaformaldehyde-Methanol FixationFix in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at –20 °C. OrPEM-Ethanol FixationFix in PEM buffer for 10 minutes. Rinse twice, briefly, with PBS. Permeabilize with cooled ethanol for 5-10 minutes at –20 °C

Fixation of fresh and frozen materialMethod of fixation has to be selected according to

1) the experimental task.

Examples: For simple identification of the protein in the cell: acetone fixation is sufficientFor precise identification of protein localization in the intracellular compartment PFA/triton is optimal

2) ability of the antibody to recognize fixed antigen.

Most of antibodies recognize antigens only in specific conditions. Example from our lab: MS-1 antibody recognizes stabilin-1 in the acetone-fixed cells, but not in PFA fixed cells

IHC and IF: overlapping termsDirect Indirect

Cheap

Fast

Advantages Disadvantages

Only limited amount of labeled primary antibodies are available commercially

Advantages Disadvantages

Takes more time, sometimes is more expensive

Additional control for the background staining is absolutely necessary

Wide range of labeled secondary antibodies are available commercially

It is always possible to design combination for double and triple staining

or enzyme

or enzyme

Controls

IHC

Background signal coming from substrate

IF

Auto fluorescence

Non-specific signal coming from secondary antibody alone

Non-specific signal coming form primary antibody.

Isotype control for monoclonal antibodies or preimmune serum for polyclonal antibodies has to be used

Experimental design for IHC

1 2 3 4

Substrate (DAB) for IHC + + + +

1st antibody _ _+ Preimmune serum for

polyclonal ab or isotype control ab for monoclonals

+

Antigen- specific

2nd antibody

_ + + +

IHC and IF: overlapping terms

Martens, Kzhyshkowska et al, J Pathology, 2006

Multuple IHC

Multiple staining can also be done with enzyme conjugated antibodies developed with different chromogen substrates to produce the end products of different colors

Identification of double positive cells by IF

Martens, Kzhyshkowska et al, J Pathology, 2006

Multiple IF

PL-FITC

Stabilin-1 staining

TGN-46 Merge

Most frequently double and triple IF are usedColor codeRed + green = yellowRed + blue = pinkGreen + blue = cyanGreen + red + blue = white

Kzhyshkowska et al, JI, 2008

Human macrophage

Human placenta

Stabilin-1

Ab CLEVER Ab F4 Merge

IHC: principle of EnVision detection system from DAKO

Enzyme: Alkaline Phosphatase (AP) or Horseradish Peroxidase (HRP)

Polymer permits binding of up to 100 HRP molecules and up to 20 antibody per backbone

Horseradish peroxidaseThe enzyme horseradish peroxidase (HRP), found in horseradish, is used extensively in molecular biology applications primarily for its ability to amplify a weak signal and increase detectability of a target molecule

In the presence of H202 (hydrogen peroxide) DAB (3,3'-Diaminobenzidine) is converted to an insoluble brown reaction product and water by the enzyme HRP

DAB + H202 ----------HRP----------> DAB ppt + H20

DAB ppt – insoluble, brown

IHC: New markers for sinusoidal cells in human lymph nodes

Martens, Kzhyshkowska et al, J Pathology, 2006

Questions?