METHODS FOR STUDYING
OF PROTEINS I
Michael Jelínek
michael.j@email.cz
CONTENT OF THE LECTURE
1) Organization of the practice from Cell and Molecular
Biology
2) General principles of specific detection of molecules
3) Microscopy techniques
4) Determination of protein concentration
5) Assessment of protein expression level
• SDS-PAGE + Western blot
1. ORGANIZATION OF THE PRACTISE
• 2 parts (2x 4 lectures)
• obligatory: for gaining credit is necessary 100% attendance and succesful passing of
the practice test (writting during the second part of practice)
• absence (due to serious reasons) can be substituted after agreement of the head of
practice by joining another studying group
• necessary equipment: lab coat, calculator
• necessary knowledge: theory from the lecture „Methods for studying of proteins I“
2 practical tasks:
1) Detection of actin and DNA in cancer cells by fluorescent microscopy
2) Comparison of protein expression in different tissues and samples by protein
electrophoresis followed by Coomassie briliant blue stainning
2) GENERAL PRINCIPLES OF SPECIFIC DETECTION OF
MOLECULES
2.1. How to find specifically the target molecule among the thousands of other
molecules? (necessary for the specifity of reaction)
2.2. How to visualize the result of the specific detection?
Most of biological structures have no colour themself... (necessary to choose the
proper system of signal detection)
2.1 SPECIFIC DETECTION OF TARGET MOLECULE
Molecule in the sample is detected via specific and known interaction
with another molecule
The molecule interacting with target molecule can be:
2.1.1 small organic molecule, which is known to bind specifically to
our target molecule (e.g. taxol binds to tubulin, phalloidin binds to
F-actin, DAPI and ethidium bromide bind to DNA)
2.1.2 protein specifically recognizing target molecule, most widely
antibody are used
2.1.1 Small organic molecule that can bind to target
molecule
DAPI, phaloidin, paclitaxel, nocodazol, ethidium bromid…
Amanita
Phaloidin
Polymerisation of actin
Yew tree
Paclitaxel
Polymerisation of tubulin
2.1.2 A. Proteins recognizing specifically cell structures
Dnase I - binding of G actin in nucleus
Concanavalin A - binding of glucose-manose groups, possible detection of
cancer cells
Annexin - binding of phosphatidylserine - detection of apoptotic cells
2.1.2 B. Proteins recognizing specifically cell structures antibodies
Most widely used system for detection of proteins uses Antibodies
= IMUNODETECTION
Antigen binding sites
Antibodies recognizing any protein can be theoretically
prepared
Imunoglobulines produced by imunne system (Ig class
A, D, E, G, M), recognize specific epitopes at the
antigene molecule
antigen = substance that induced production of
antibodies directed to it by immune system, (usually a
foreign molecule)
epitope = specific area of the antigen surface that is
bound by the antigen binding sites (part of antibody)
Light chain
Haevy chain
2.2. POSSIBILITIES OF SIGNAL DETECTION
Detecting molecule (specifically recognizing target molecule) is usually not
possible to be directly observed (exception e.g. DAPI)
Thus, detecting molecule must be conjugated with another molecule that is
able to produce detectable signal
Such conjugates can be:
2.2.1. Heavy metal
2.2.2. Fluorophore
2.2.3. Enzyme whose enzymatic activity enable visualization of detected
molecule - light, colour signal
Direct and indirect (immuno)detection
Labeled primary antibody (direct detection)
vs
Non-labeled primary and labeled secondary antibody (indirect detection)
Direct detection
Indirect detection
detectable
product
detectable
product
substrate
substrate
Signal amplification
enzyme
enzyme
secondary antibody
primary antibody
2.2.1 Vizualisation by conjugated heavy metal
Production of somatostatin in pancreatic
cells
2.2.2 Vizualisation by conjugated fluorophore
fluorophore = molecule that is able to absorb light of a specific
wavelength and emits light at a longer wavelength (=signal we detect)
2.2.3. Visualization by the activity of conjugated enzyme
Visualization of target molecule after reaction of enzyme with its substrate
chemiluminiscence - enzyme horse radish peroxidase (= HRP) is usually used. HRP cleaves
hydroxide peroxide and created radicals activate luminol - light is emited and detected (by
special instrument or photography film e. g. western blot)
chromogenic (color reaction) - enzyme produces coloured product (colorimetr, e.g. ELISA)
• product must be insoluble if the localization
of
target
molecule
is
detected
imunofluorescence
• product must be soluble if intensity of the
colour in solution is measured (quantification
of the target molecule via measurement of
absorbance)
3) MICROSCOPY TECHNIQUES
3.1 Imunohistochemistry
3.2 Electron mikroscopy
3.3 Fluorescence microscopy
3.1. IMUNOHISTOCHEMISTRY
detection in microscopy preparat (thin tissue section) often used
usually specific antibodies used and chromogenic reaction or fluorescence show
localisation of the target protein
Insulin in cells of pancreas
3.2. ELECTRON MICROSCOPY
Not so often used
Somatostatin in D cells in pancreas
3.3. FLUORESCENCE MICROSCOPY
Using of single fluorophore vs using of more fluorophores
Using of various fluorophores - possibility of observation of more types of molecules as well
as their colocalisation (presence on the same cells or at the same place in the cell)
At the practise: actin and DNA stainning
The signal only from one fluorophore can be observed in a microscope, for multicolour
stainning multicolour picture must be assembled from single colored pictures by specific
software
 DNA: stained by DAPI
 tubulin: stained by anti-tubülin antibody conjugated with fluorophore FITC
 actin: stained by phalloidin conjugated with fluorophore TRITC
4) DETERMINATION OF THE CONCENTRATION OF
PROTEINS IN SOLUTION
based on spectrophotometry (measurement of absorbance)
4.1 Determination from absorbance in UV spectrum
4.2 BCA assay (Bicinchonic assay)
4.3 Bradford assay (will be used in practice)
4.1. Determination from absorbation in UV spectrum
Proteins naturally exhibit absorption in UV part (260 - 280 nm) of spectrum
- primarily due to presence of aromatic aminoacids
(tyrosine and tryptophan)
no need for calibration curve
Calculation: [Protein] (mg/mL) = 1.55*A280 - 0.76*A260
!!! all methods that are based on detection of presence of only certain types of
aminoacids in samples are reliable only if there is no big difference in aminoacid
composition in proteins of analyzed samples !!!
4.2. BCA assay
Detection reagent contains BCA (bicinchoninic acid)
Colorimetric reaction results from interaction of BCA with peptide bond
(and not only from interaction with specific aminoacids)
Intensity of purple colour is measured at 562 nm
Protein concentration
4.3. BREDFORD ASSAY
Principle: colorimetric reaction after mixing of Bradford reagent with proteins containing
solution
Bradford reagent contains Coomassie Brilliant Blue - binds to basic and aromatic AA in
proteins (Arg, Phe, Try, Pro)
Presence of proteins changes the colour of solution from brown to blue
Absorbance is measured at 595 nm (absorbance correlated with concentration of proteins)
Protein concentration
QUANTIFICATION OF PROTEINS IN SAMPLES
construction of calibration curve using samples with KNOWN
concentration of protein - usually serial dilution of bovine serum
albumin (BSA)
Measured value of absorbance
Corresponding protein
concentration
5) ASSESSMENT OF PROTEIN EXPRESSION LEVEL
Metods based only on immunodetection: ELISA, flow cytometry
SDS-PAGE + Western blot
SDS-PAGE - Method used for separation of proteins according to their
molecular weight
= sodium dodecyl sulphate polyacrylamide gel electrophoresis
used for determination (comparison) of protein expression in samples
sample in the form of protein-containing solution, e.g. tissue lysate, …
before analysis, desintegration of cells (= cell lysis) is necessary to release the
cell content into solution
cell lysis is performed usually by various chemical, mechanical and physical
approaches, or by their combination
5.1. Preparation of samples
for separation, proteins must be denaturated into individual polypeptide chains
Proteins must be denaturated by denaturatig agens before separation
SDS (sodium dodecyl sulfate) wih heating are used releasing proteins into fibre
form
merkaptoethanol, dithiotreitol - reduce S-S bridges
They also have homogenic negative charge due to SDS
treatment, the same for length unit of protein
5.2 Principle of electrophoresis
gel electrophoresis  gel with such pore
size that enables molecules with different
size to move with different speed
proteins: usually polyacrylamide gel
(electrophoresis of DNA, RNA: usually
agarose gel - bigger pore size than
acrylamide gel)
5.3 Course of electrophoresis
negatively charged proteins move to positive electrode,
longer polypeptides chains are more retarded by the gel structure than shorter
chains
 shorter chains travel faster
Separation of proteins only according to molecular weights, proteins are
negatively charged - they move in electric field to anode - positive electrode
molecular weight of protein of our
interest can be found by
comparing its size with molecular
weight marker = commercially
available mixture of proteins with
known molecular masses
5.4 Gel stainning
Blue stain Coomassie blue binds
nespecifically all proteins (see
Bredford method), each strip (band)
represents a group of proteins with
certain size
Marker Increasing concentration of
protein
5.5. Western blot
SOUTHERN blot (technique for DNA detection)
developed by Edwine Southern (1975)
NORTHERN blot (technique for RNA detection)
WESTERN blot: Used usually for comparison of
protein expression using immunodetection
Transfer of proteins separated by SDSPAGE to a protein-binding surface
(nitrocelulose or PVDF membrane)
negatively charged proteins move from
polyacrylamide gel to the membrane
membrane enables easy manipulation and
easy access of antibodies during next steps
of the target protein detection
proteins stick to the membrane in the same position they had in the gel
membrane blocking (usually BSA or defatted milk) - saturation of all protein binding-sites on the
membrane
detection of target protein by specific antibodies
detection of protein of our interest usually by specific antibodies and chemiluminiscent or
chromogenic reaction with formation of insoluble product
5.6. Medicinal application
Membrane strips containing electrophoretically
separated antigen extracts are used as solid
phase.
The position of the proteins depends on their
respective molecular masses.
• If the pacient´s sample is positive, specific
antibodies in his serum attach to the antigens
coupled to the membrane.
• The attached antibodies react with AP-labelled
anti-human
antibodies
(AP
=
alkaline
phosphatase).
•The enzyme activity than mark the sites where
is the antibody bound (if it really is),
presence of the antigen in
blood of pacient is finally
proved/disproved
Detection of antibodies (e.g. for confirmation of diagnosis)
• boreliosis, EBV, HIV, HSV, Helicobacter pylori
• autoantibodies, antibodies against nuclear antigens (ANA),
antibodies against neural antigens