ALP (alkaline phosphatase) calibrators were analyzed manually in microtiter plates to find the linearity range by following this protocol:

Transcription

1 Exam Mol 3008 May 2009 Subject 1 (15p) ALP (alkaline phosphatase) calibrators were analyzed manually in microtiter plates to find the linearity range by following this protocol: Reaction solutions: 50 μl of calibrator/control/sample is added to each microtiter plate well, duplicates. 50 μl of buffer-substrate reagent is added to all wells. Start the timer when adding reagent to the first well. Cover the wells with parafilm, and put the microtiter plate into a 37 C incubator. Incubate for 30 minutes. 200 μl 0.2 M NaOH is added to each well in the same order as adding buffer-substrate reagent. Blanking solutions: Add these solutions in a new microtiter plates in following order: o 200 μl NaOH o 50 μl calibrator/control/sample o 50 μl buffer-substrate reagent No incubation. Read the absorbance in a microtiter plate reader, at 405 nm. Subtract the blank absorbance from the reaction absorbance. Find the calibration factor/calibration curve, and calculate the ALP results. This table shows the absorbance results of the calibrators: Calibrator concentrations, ALP Absorbance (average of duplicates) Blank solution Absorbance (average of duplicates) Reaction solution 0 U/l U/l U/l U/l U/l U/l Find the linear range for this analysis. Draw a diagram, and explain (mm paper not necessary). What would you do if some of your samples were measured above the linearity range for the method? What change(s) would you do to the method if all your sample results were measured < 1.5 U/l to improve the sensitivity? 1

2 Subject 2 (15p) a) Describe the technique called western blot or immuno-blotting. Suggest strategies for how to ensure that the signal you get represents the correct protein(s). b) You would like to visualize the localization of two different proteins (antigen) simultaneously by immuno-staining. What considerations would you have to do previous to staining? What kind of controls could you do to ensure the specificity of your signal? c) Antibodies are important tools in life science. - What is the difference between a primary and secondary antibody? - What is the difference between a native and denatured antigen? Subject 3 (10p) a) Outline schematically the most important parts in equipment used for High Performance Liquid Chromatography (HPLC). b) If your chromatogram has peaks that are not well separated from each other, how can you improve the resolution in a HPLC analysis? Subject 4 (10p) a) Describe the fundamental principle of a confocal laser scanning microscope (confocal microscope). b) Discuss briefly the need of controls and relevant source of errors. Subject 5 (5p) Describe a simple enzyme-linked immunosorbent assay (ELISA). Describe how such an assay now can be used to determine the amount of several antigens simultaneously in the same tube. Subject 6 (5p) When measuring protein concentrations with BioRad Protein Assay, the linearity range of the method depends on what standard (calibrator) material you choose; an albumin standard or a globulin standard. Explain why. 2

3 Multiple choice questions (only one cross for correct answer). Correct answer gives 1 p 1. Quantification of immunoreactive proteins may be performed by turbidity measurements. Turbidity causes light scattering which involves A B C D A restricted range where Beer`s law could be applied Increased emission Reduced transmission Reduced emision which should be taken into account while calculating the results 2. A competitive inhibitor competes with the substrate in binding to the enzyme. This kind of inhibition causes A Decreased Vmax and increased Km B Increased Vmax and unchanged Km C Decreased Km and unchanged Vmax D Increased Km and unchanged Vmax 3. A non-competitive inhibitor binds to the enzyme but does not interfere with the catalytic site of the enzyme. This kind of inhibition A B C D Is always irreversible Is due to changes in configuration of the protein Is due to changes in conformation of the protein Is caused by a prostetic group 4. In an electrophoresis system, using a native gel (e.g. agarose), proteins are separated due to differences in total charge. What decides the proteins total charge? A The voltage B The buffer s ph C The protein size D The support medium 5. In electrophoretic separation of proteins in an agarose gel, electroendosmotic flow occurs. This is a buffer flow that may disturb protein migration in the gel. Which of the following alternatives will reduce electroendosmotic flow? A Applying less amount of sample B Increasing the voltage C Running the gel at low temperature D Using a support medium with lower charge 3

4 6. The image shows serum proteins separated in 1% agarose gel, 25 minutes at 100 Volts, stained with Ponseau S protein stain. The separation of the proteins is not optimal. + What would you do to improve the separation? A Increase the agarose concentration B Run the electrophoresis at 75 V instead of 100 C Run the electrophoresis for 15 minutes instead of 25 D Use a lipid stain instead of a protein stain 7. The schematic drawing shows separation of DNA fragments in a capillary electrophoresis system, where the capillary is filled with a polymer. The separation is based upon the following principle: A Differences in current during the electrophoresis run B Different charge of the DNA fragments C Different molecule size of the DNA fragments D Differences in electroendosmotic flow during the electrophoresis run 8. Chromatography is often divided into gas chromatography and liquid chromatography. For gas chromatography, which is the most correct characteristic: The mobile phase is a gas and A the stationary phase is a liquid B the stationary phase is a solid C the stationary phase is a liquid or a solid D the samples to be separated are gases E the samples to be separated are liquids 4

5 9. Chromatography can be used to estimate the size of a molecule. Which chromatographic principle is used for this purpose A Partition B Affinity C Gel (SEC) D Chiral E Ion exchange 10. Chromatography can be divided into planar and column chromatography. Planar chromatography is most correctly characterized by A The mobile phase is a gas or a liquid and the stationary phase is a liquid or a solid B The mobile phase is a liquid and the stationary phase is a liquid or a solid C The mobile phase is a liquid and the stationary phase is a solid D The mobile phase is a liquid and the stationary phase is a liquid E The mobile phase is a gas or a liquid and the sample is a liquid 11. In Chromatography a highly efficient system is appreciated. An efficient column is most correctly characterized as a column that can: A Separate compounds with short retention times B Separate compounds of large volumes C Produce large amount of results D Separate compounds with very similar properties E Separate compounds with very different properties 12. Chromatography can be used to separate, identify and quantify components in a mixture. You have an HPLC analysis with UV-detection Can you estimate the most abundant components in the sample? A Yes, the peak with the highest top is the most abundant component in the mixture B Yes, the peak with the largest area is the most abundant component in the mixture C Yes, the peak at the highest retention time is the most abundant component D No, the peak size in a chromatogram can not be compared with each other for quantification E No, the peak size does not reflect the amount of the component in a sample mixture 13. Mass spectrometry (MS) is a very powerful technique Which is the most correct characterization of an MS detector? The MS detector: A Separate compounds according to their molecular weight B Separate compounds according to their charge C Separate compounds according to their boiling point D Separate compounds according to their mass and charge E Separate compounds according to their mass 5

6 14. Ion exchange chromatography is widely used in biochemical analysis Which is most correct; Ion exchange is used to analyse A Inorganic ions B Organic ions C Metal ions D Cells and proteins and aminoacids E Most molecules that form ions 15. Ion selective electrodes (ISE) are widely used in hospital laboratory analysis Which is most correct; ISEs are used to analyse A Inorganic ions B Organic ions C Metal ions D Cells and oxygen and ions E Oxygen and inorganic ions 16. A laser scanning (confocal) microscope allows us to obtain optical slices of the imaged sample. What particular component(s) is crucial for this? A Lasers of different wavelengths B The pinhole C Fluorescent dyes with different emission wavelength D The digital detector(s) E Optical filters to discriminate excitation and emmision light 17. Fluorescent detection is based on the Stoke`s shift of the fluorochrome What does the Stoke`s shift describe? A Difference in emission wavelength for different fluorochromes B The emission light intensity of different fluorochromes C The exitation/absorbtion maximum wavelength for different fluorochromes D The difference between exitaion and emmision wavelength of a fluorochrome E The fluorescent stability of a particular fluorochrome 18. Auto-fluorescence can be a limiting factor in fluorescent imaging. What is causing auto-fluorescence? A Unspecific binding of the primary antibody B Incomplete washing/removal of the added fluorochrome C The dye give rise to fluorescence without exitation D The emitted light from one fluororescent dye exites the second used dye E Endogenous proteins in the tissue/cells with fluorescent properties 6

7 19. Green fluorescent protein (GFP) has become an important tool in life science. What property of GFP is particularly useful? A The high enzymatic activity that allows sensitive detection B The high photo-stability C The fluorescent property of GFP fusion proteins D The high fluorescent intensity E The large Stoke`s shift 20. Fluorescent imaging can be quantified in terms of differences in relative intensity What is this quantification based on? A Relative intensity detected by the grey-scale detector(s) B The absolute intensity of the particular fluorochrome C The colour of the fluorescent dye D The relative intensity detected by the digital colour camera E The large Stoke`s shift 21. Luciferase is used extensively in many different approaches in life science. What is luciferase? A A fluorescent protein B A fluorescent dye C A fluorescently labelled secondary antibody D An enzyme E A reporter plasmid 22. Monoclonal antibodies are important tools in many aspects of life science research and diagnostics What is a monoclonal antibody? A An antibody made against a short peptide B An antibody that can bind only one antigen epitope C An antibody that recognize either a native or a denatured antigen D An antibody made in mice E An antibody made by one clone of B-cells 23. Fluorescence recovery after photo-bleaching (FRAP) is a technique to study protein dynamics in live cells. What is this technique based on? A The irreversible photo-bleaching of a fluorescent protein B The reversible photo-bleaching of a fluorescent protein C The intensity of green-fluorescent protein (GFP) D The mobility of a particular fusion protein E Rapid image acquisition of live cells 7

8 24. A flow-cytometer can be used to detect the labelling intensity of several fluorochromes simultaneously. What properties of the fluorochromes are particularly important in such a setting? A The photo-stability of the fluorochromes B Differences in emission/absorption maximum C The different dyes stain different cell surface markers D Differences in emission spectra E The different dyes have similar fluorescent intensity 25. Enzyme cascades are common biology, e.g. the product in an initial reaction constitutes the substrate in the next reaction. This is often utilized in determination of enzyme activity in vitro. In the following cascade product D is a chromophore. What experimental conditions are mandatory to determine the enzyme activity of E1? A + E1 AE1 B + E1 B + E2 BE2 C + E2 C + E3 CE3 D + E3 A Excess amount of A, E2 and E3 B Excess amount of B, E2 and E3 C Reaction solution with coenzyme D Addition of metal ion E Excess amount of all substrates 26. Enzymes are biochemical catalysts Which describes the primary feature of enzymes? A They are covalent bound to metal ions (e.g. Mg2+) B They lower the activation energy of a reaction C They decrease the rate of the reaction D They bind to a variety of substrates E They reduce Km 27. Enzymes possess regulatory and active sites. What characterize the active site? A Nonadjacent amino acids can make up the active site B The active site always consist of a single region that binds the substrate and catalyses the reaction C The amino terminus of the protein always contains the active site D The active site interacts with the substrate through a peptide bond 28. Enzyme variants (isoenzymes) may occur in a different organ or different cell type Isoenzymes are proteins that A Have arised from point mutations B Differ in amino acid sequence C Are often identified by microarray screening D Display similar immunochemical features 8

9 29. DNA and RNA quantity is assessed by photometric measurements at 260 nm Additional measurements at 280 nm is performed A To distinguish DNA from RNA B To calculate the molar absorbtivity (extinction coefficient) C To determine protein concentration D To calculate your DNA/RNA ratio 30. In molecular cell biology we often use reportergene studies. These studies are used to A Demonstrate localization of genes B Demonstrate localization of proteins C Examine transcriptional activation of genes D Examine protein-protein interactions 9