Michael Jelínek michael.j@email.cz METHODS FOR STUDYING OF PROTEINS I Michael Jelínek michael.j@email.cz

CONTENT OF THE LECTURE Organization of the practice from Cell and Molecular Biology General principles of specific detection of molecules Microscopy techniques Determination of protein concentration 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: 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… Phaloidin Amanita 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) substrate enzyme detectable product Direct detection Indirect detection Signal amplification 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

detection in microscopy preparat (thin tissue section) often used 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

Somatostatin in D cells in 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 Marker Increasing concentration of protein Blue stain Coomassie blue binds nespecifically all proteins (see Bredford method), each strip (band) represents a group of proteins with certain size

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 SDS-PAGE 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