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BE 484 Analytical Methods in BiotechnologyMajor Exam (2ndsemester 2003-2004)

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Theory Max Marks: 40Time: Total 2 hours

Q. I What is the basic principle of chromatographic method? Explain the differencebetween open and closed column chtomatography. Between HPLC and FPLC, whichmethod has high resolving power and why? I + 1 + 2 = 4

Q. 2 In a protein there are two solvent accessible tryptophan residues, one is on thesurface and the other is in the interior surrounded by charged residues. How do youselectively quench the tryptophan on the surface?Explainwith principle the experiment.2

Q. 3 State and define various parameters that are used to evaluate the performance inchromu~0graphicseparations. 2

Q. 4 Distinguish between g:-oupspecific ligand affinity chromatography and immobilized'metal affinity chromatography. Illustrate your answer with suitable examples. 3

Q. 5 How does the polarity of the solvent affect the absorrtiuD maxima of the protein?How can the solvent rJerturbationmethod be' used to find the position of a particularan1inoacid in native protein? 3

Q. b a. What is the principle on which zonal and isoUichl'phoresis electrophoreticsystems are based? Gi"r. an application for each category. 3

t... What is the function of the stacking and C separating gel 4\ystemsin DISCelectrophoresis? .2

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Laboratory Questions Max. Marks: 20

I Q.1 a. While checking the punty nf a protein, which chromatographic system (FPLC orI HPLC) would you prefer and why? 2

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b. Proteins A and B interact in aqueousmedium. How can you show with the HPLCexperiment that they are binding to each other. Explain with thc drawings of thechromatogram. 2

c. You have bC0ngiven two different concentrations of a pure protein for recordingCD spectra. Would you get same results while caJculating its secondary structure? I

d.What is the need for having a pure protein in CD studies? 2

Q.2 a. Why is methylation of a triglyceridecarried out prior to GC analysis?

b. State the reaction calalyzed by phospolipaseA2. ...

c.What is the principle of Folin-Lowrymethod for estimation of proteins in afermentation (cell-free) broth?

d. Distinguish between transmittance andabsorbance?2

e. Name different parts of a typical doublebeam UY/visible spectrophotometer.2

Q.3 a. Proteins A and B having a molecular weight of 16,500 and 35,400 move 1.3 cm

and 4.6 cm, respectively. whcn clecfrophore~edthrough a gcL What is thc molecularweigh~of a protein C, which moves 2.8 cm in the saplp,gel? 2

b. Wny is electrophoresis dOI1P;0 solutions of low sait concentration?

c. A -,mall enzyme has a sedimentation coefficient:Jf 3.4 S. Whcn it bindS'to itssubstrate (a small organic molecule), its s value changes to 2.9 S. Explain this change. I

d. If a boundary moves halfway down a centri(uge cell in 20 min at 20,000 rpm,how long w',:Jit take to reach the same position if the speed ;s 40.000 rpm ? 2

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d. Show the ions being formedwith methaneas a reagentgas. Wouldthese becalledas the primaryor secondaryionsandwhy?Whatis theirfunction? 2+ 1+ 1 = 4

Q.8 What is the difference between boundary and zonal sedimentation and under which

!conditions(for what kind 0f data generation)would you use these two methods?; 2+1= 3

i'Q.9 a. The specific activity of a sample ofe2p] ATP is 5.3 Ci/mmol on Jan 23. What willthe specific activity be on Feb 18of the sameyear~given the half-life of32p is 14.2days?

b. In an experiment to measure DNA synthesis, would you use eH] thymidine ore2p] nucleotide and why? 1

c. You have in your lab a Geiger counter with an efficiency of 22% for 14Cand a

background of 6 cpm and a scintillation counter with an efficiency of 72% for 14Cand abackground of 38 cpm. In an experiment with 14(:,you expect your sample to have verylow activity-that is, from 75 to 100 cpm.Which counter should you chose andwhy? -3

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