Practical molecular biology: PROTEINS

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Practical molecular biology PROTEINS

• Dr. Alexei Gratchev
• PD Dr. Julia Kzhyshkowska

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Literature

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

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Protein analysis Immunohistochemistry (IHC)

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

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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

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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-(Aß-)peptides
3. Accumulation of APP-sb results in the
formation of plaque in the brain
1. Amyloide-precursor protein (APP) is
constitutively exposed on the surface of a
neuron. Cleavage of APP is performed by
a-secretase results in the production of soluble
APP-sa
2. Internalisation of APP results in the
pathological intracellular APP processingand
secretion of APP-sb
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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

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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

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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)
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Material for IHC and IF

• Paraffin embedded
• Tissue sections

• Fresh or frozen
• Tissue sections
• Cells grown on cover slips
• Cells sedimented on object glass
• using cytospin centrifuge

Advantages
Disadvantages
Advantages
Disadvantages
Limited time of storage Retrospective analysis
is not possible
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
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Fixation of fresh and frozen material
Method 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 sufficient For 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

• Methanol-Acetone Fixation
• Fix in cooled methanol, 10 minutes at 20 C.
• Remove excess methanol.
• Permeabilize with cooled acetone for 1 minute at
  20 C.
• Or
• Paraformaldehyde-Triton Fixation
• Fix in 3-4 paraformaldehyde for 10-20 minutes.
• Rinse briefly with PBS.
• Permeabilize with 0.5 Triton X-100 for 2-10
  minutes.
• Or
• Paraformaldehyde-Methanol Fixation
• Fix in 3-4 paraformaldehyde for 10-20 minutes.
• Rinse briefly with PBS.
• Permeabilize with cooled methanol for 5-10
  minutes at 20 C.
• Or
• PEM-Ethanol Fixation
• Fix in PEM buffer for 10 minutes.
• Rinse twice, briefly, with PBS.
• Permeabilize with cooled ethanol for 5-10 minutes
  at 20 C

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IHC and IF overlapping terms
Direct
Indirect
Disadvantages
Advantages
Disadvantages
Advantages
Cheap Fast
Only limited amount of labeled primary
antibodies are available commercially
Wide range of labeled secondary antibodies are
available commercially It is always possible to
design combination for double and triple staining
Takes more time, sometimes is more
expensive Additional control for the background
staining is absolutely necessary
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Controls
IHC
IF
Background signal coming from substrate
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
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Experimental design for IHC
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IHC and IF overlapping terms
Martens, Kzhyshkowska et al, J Pathology, 2006
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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
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Identification of double positive cells by IF
Martens, Kzhyshkowska et al, J Pathology, 2006
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Multiple IF
Ab CLEVER
Ab F4
Merge
Human placenta
Stabilin-1 staining
PL-FITC
TGN-46
Merge
Stabilin-1
Human macrophage
Color code Red green yellow Red blue
pink Green blue cyan Green red blue
white
Most frequently double and triple IF are used
Kzhyshkowska et al, JI, 2008
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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
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Horseradish peroxidase

• The 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----------gt
DAB ppt H20 DAB ppt insoluble, brown
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IHC New markers for sinusoidal cells in human
lymph nodes
Martens, Kzhyshkowska et al, J Pathology, 2006
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Questions?