8/14/2019 Bel420 Major Sem2 08

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Time: 2 hours

;;?7 fPBEL420 Analytical Methods in Bi nology ,/] /!Major Exam Hnd semeste 2007-08 iI -c;C,/J/I( IvIJ.c--------'30- 0'1 - 2oD 8'

I:::v:LJ.J'DtJ jT fV).Max Marks: 50

1. A key and often laborious element in analytical methods is the eliminationof/correction for errors. In isotope exchange gas chromatography coupled to massspectrometry analysis of a model biological system, list possible elTors and difficultiesand eXDlain how they are corrected/overcome (in tabular form). (12),/

Explain the principle of fluorescence microscopy. (6),/'. h) An analysis of cell contents shows their cytoplasm contains 5% by weight oflutes: 1% is proteins of average molecular weight 45000; 1% is soluble lipids of

m~ecular weight ~ I% is sugars of mole~u'-ar weight 170~nd 2% is salts like !SCt.What is the osmotic pressure TfiSide these cells relative ~ure watd at j t"C? If tEeintracellular contents are released in a more controlled way (e.g. by a press) rather thanby simple osmotic shock as above, what would a typical intracellular release vs. numberof passes profile look like? Show by a sketch. (6)+(6)

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3. Two proteins have the same molecular weights. At pH 5.5 both have considerablesecondary structure and are at least 75% a helix. At pH 8.5, one of them loses allstructure and is a random coil; when returned to pH 5.5, its structure is restored. Devise aprocedure for separating these proteins. (3)

)f A procedure has been devised for purifying 1t particular enzyme using gel. . chromatography. In an effort to increase the amount of material to be handled, you use as pie whose concentration of protein is ten times that normally used with thisprocedure. The enzyme activity now elutes principally in the void volume. Explain whathappened. .In general, to increase the amount of material to be handled, what physical parameters ofthe column should one vary? (4)

~ J'Write five most important empirical rules of interpreting fluorescent spectra of~ns. (3)

;J2' (i) How ma~y ~pm are a~so~iatedwith 1yCi ?~radioa~tive material? lfthe instrume~t.used for countmg IS25% efficIent at detectmg dlSlntegratlOnevents, how many cpm w1l1i yield? What is Cherenkov counting and for which radioactive material can it beu~? 0r

(ii) What is the role of SDS and polyacryamide gel in SDS-P AGE electrophoresis?What is the use of Fergueson's plot? (2+ 1)r-

(iii) Are there matrix peaks in the electrospray mass spectra and in MALDI? Explain.Name two matrices used and their properties. (3)~

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