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"-- ~BEL 420: Analytica' Methods in BiotechnologyMajor Test IInd Semester' 2006-2007i1ax.Marks: 40Part B (15 Marks)

1. (a). It was observed that thc purification of a recombinant protein is difficult usingsizc exclusion chrorl1atography,on exchangechromatography and hydrophobicinteraction chromatography. What can be done at any level like chromatography,genetic manipulation etc or both to get sufficient a111ountof that purifiedrecombinant protein? Explain with merits and demerits of your proposition. (5)(b). What kind of consid~ratiol1sneed to be takcn for the prepnrationand

, purificationof recombinant proteins to be used in different purposes like humanI healthcar~,industrialpurposes,structuralstudiesetc.(5)

(c ). A protein preparation is exhibiting substantial secondary structure from Far-UV CD spectroscopy. However, the near-UV CD spectroscopy is not snowing theexistence of tertiary structure. Although the X-ray diffraction data confirms thcpresence of tertiary contacts. What could be the reason for this and how are you goirt'gto trouble shoot the problem. (5) \

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BEL420 Analyticall\1cthods in Biotcchnolog}'Major Exam 2ndsemester 2006-07. @Time: 2 hoursf..1ax.Marks:40I Part A (25 Marks)

Qt 1 a. Examine the electrospray spectrum of phage lysozyme. Determine the mol wt ofthis protein. ,-.- 217-

11}' 10119.8991.5GO'v,~ 80''Vit.:Q):s 60' 19'939.2

16-1115.515+1189.6

t. 14-1274. 13- 12-':1372.5 1406.6'. I

~,J~~l!!ln"~1200 14~O 16~)miz800 1000

I b.Distinguish between FAS and MALOI assourcesof ionization ofbiomoleculcs.Highlight the advantages/disadvantages of thesetechniques. 2 + 2 = 4I . ,Q.2 a.DefineoneCurie (Ci).How is it relatedto disintegrationperminute? 2b. Why are fiuors added to the counting liquids in liquid scintillation counting? Whatis the difference between a primary and a sccondary fluor? 4c. Good way to deco'ntaminate 32pcontaminated glassware is to store it in leadcontainers. How long should glassware contaminated with 2 x'10('cpm be stored so thairadioactivity is no more than 100cpm? Could storage be used to decontaminateglassware containing 3H (Half.life, 32p=l4.2 days; 3H=12.3years) 3d. A mutant of E. coli has the property of dividing asymrr.etrically,and producjng anormal cell and one having approx 1/101hvolume at a high frequency. Jhe s111allellsnever divide again. How could you determine whether the small cells have the normalamount of DNA? I 3

Q.3 A stretch of DNA is beingsequencedas shownbelowbySanger'smethod.Showthelength of the reaction products on the 4-lane (A,C,G,T) gel.5' -CCGA TCT ATACCA T-3'GGTA-5'

JA a. What is the difference betw.eensedil11e~tation-velocityand sedimentation~l1\hbnUm method? Wnte a practtcal appllcatton for each. 3b.2-D liquid chromatography is beiilg used to achieve good resolution of proteins.'"What principles, in your opinion, can be employed in this methodology? 2

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