BIBC 103 - exam 2 - toukdarian

BIBC 103 - exam 2 - toukdarian - “if I (Y UL.- ' balsa-ow...

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Unformatted text preview: “if I (Y UL.- ' balsa-ow 103 (,0; I , Exam 2 Section: name. Winter 200'? 5 pages -- Write legibly. You may use the back of the page for an answer if necessary, but indicate you are doing so. 1) (20 pts) Fill—in the blanks: is 11) C) d) g) a) Antibodies are made by a class ofwhile blood cells called 8’ Ce l {f Each lgG antibody molecule is a tetramer composed of two identical L‘Edv I elm/n r and two identical {I‘filtf g' Ltd {/1 f . Provided there is no steric hindrance, the number of antigen molecules that one antibody molecule can bind to is W O The part of an antigen that the antibody binds to is called the ‘8 @1310 g f 1 . Two e1 mes commonly used to label antibodies are horseradish peroxidase and dill/Ca (N, E l’tofglw/L 7L“, /~€. PM _ i v“ [ Cortisol is produced by the gztnd in response to f " Eff. MAPK is an example of a protein WhOSe activity is regulated by phosphorylation. in an unfertilized sea urchin egg the Elm/glww'glmlv'kl form of MAPK is the active form. The role of active MAPK in an unfertilized Sea urchin egg is to i M {1.510; é DNA replication. 2) (12 pts) On the following diagram of an SDS—PAGE gel label the two parts ofthe gel and in one Sentence describe the function of each part. 7/ [fitted/fie «— yiaclti'wy ("a/*Wffid 7g GWhalers/r: #e M <— Furl/tiny + prank MI is} Wdecmlar Mlyé'th. b) The Electrophoresis Buffer used to run an SDS-PAGE gel has a different pH then the buffer used to pour the “upper” gel. What is the approximate pH of the Electrophoresis Buffer and the approximate pH of the “upper” gel buffer? ’7 pH of Electrophoresis Buffer: 3 pH of buffer used to pour the “upper” gel: 6 '- 00 c) Why is this pH change important? (Be specific) to 4m Clof€ felt” W Fl‘l Cl’L‘WIyJa-x 6.4:“ I Lip/63V gel {oméfew ,‘f Mm; . fiUCFfl-fl, in +lme, flecks“? { 4'- f {713' fig 4% 9/}C'vme (tl‘fgpl “fl Bulb-LC ‘ ,LLM EMA khan/you “J (/1 flat e Vic/eat» 4e refit/4,718 71%. +0 {if pi. m 1 W” any +N'fioini30 I ) “if/i,ny a {an/ff Local Vet-hiya 4,-ééew9406,#g Angel-an“ aka a 2) LI . fa, .. Section: C '0‘ Exam 2 Winter 200?' 3) (4 pt) What are the two functions of SDS in the protein sample buffer used for SDS-PAGE? {Of CL-QAA'ILWW'C/ dike fwd-eff) O'F [71/ fLL it’d-e A Vkrvi Cort/7"”; 4 M [a Vokr‘fl w .«1/'% flay/rt 1912, C ha,— f6. _ 4) (3 pt) If you Were working with a l 50 kDa protein and wanted to improve its separation from similarly sized proteins on an SDS-PAGE gel you should: (choose one) Choose a gel with a high concentration of acrylamide in the running gel. ) Choose a gel with a high concentration of acrylamidc in the stacking gel. 0) Increase the concentration of 3138 in the Electrophoresis buffer. d Heat the sample for l min instead of2 min prior to loading. 9) None of the above. 5) (6 pts) The TBST + BSA blocking solution we used for Western Blotting has the following composition: 10 mM Tris-HCI pH 7.4 150 mM NaCl 0.1 % w’v Tween-20 5 % BSA :1) What is the function ofthe Tween-20 in the TBST + BSA blocking solution? (Be specific.) TM TWAV’LQ ‘I‘D @VI*'/‘/-fl—ecf‘ - Fc; Rim“ WW lid—Ww lam-Jen; (Him 7PM furyij b) What would happen ifyou forgot to add the BSA to the in the TBST + BSA blocking solution? (Be specific.) , _ w final/J VLVL/m .ovt7l’ v—e ((J/rfilf H ’1 “J T ioio‘i" Cifm/ becijf-E 44,"; wamtak M‘JF 7g Lia/95% f‘vnrffl‘fibéz‘aé'c fgijg’tcimn [Marga “Mi, main rtst Whee- arm I ~- 6) (3 pt) Ifthe protocol you are following for a Western blot says to use a 1:1000 dilution of the primary antibody in TBSTBSA, how much of the primary antibody would you need to add to 15 ml TBSTKBSA? (Show your work for full credit.) W : X A Q ioov gaffe [In/ti rng/flf/yl 1 o .90 X _—: I ,1 it .63 d {INA/)1 «it x h/ 7) (4 pt) Why is methanol added to the transfer buffer used in Vestcm otting? #fMoL/Q/ flawt JIM flhfir‘fl I/‘Qwaf’i’wlofc’ J’IvLBJ’ +1447L (a A Err-oi 7‘7: JUL; feL—f":/ . “BRA—ye, "Fl/4nka WMQFAW.E§ whet; karat—WW io—flmljc-—-+.._6.L&a<.rf who A rye-t n fi'Lg/we/A Oar io‘d‘WW. 2 Points; i 3) 607/ Exam 2 Section: Winter 2007 Name: _' 8) (16 pts) Thyroid Stimulating Hormone (TSI-I) is a glycoprotein hormone secreted by the pituitary gland. It is composed oftwo subunits, o: and B. The ct subunit is similar to tt subunits found in other hormones (Ll-I, FSI—I and hCG) but the [3 subunit is specific to TSH. The following reagents are part of a commercially available kit to measure T SH: 10 microwells coated with anti-[3 TSI—l monoclonal Ab anti-o. TSI-l Ab conjugated to horseradish peroxidase TMB substrate wash solution Stop solution '3' TSH standards each at a different concentration ‘ ' '-. «Ma‘s-Ik- a) What iS a monoclonal antibody? . (SJ-3 3 A Wrtoziqflni Amij- 0”” 0% 06186591 AnigenlL/efl,3Lbfle 1'14 (6” but L4 1%. Lola-3V A (£11579! 71444” W347 6'9"“ "A’lflh-‘Ji'ff. I 3L r7 m.‘ :3 _ ra-l-f tuber”?! It If ditch“ cit-24 “*4 [ewe-J +7 ."‘ (“WP C?" Wis-8,11“ t"? c an gym-«4.2.1 fad»? {t'qJ-Et')’ [gram 6L. (4’6 b) he measurement of 1 SH levels In serum 15 used to chagnose thyrord disorders. Brie y ' describe how you would perform an ELISA using the reagents listed above and a serum sample from Patient A in order to determine the level of TS H in the patient’s serum. (You can list the steps you would follow or make a flow diagram. Your answer should fit in the space provided). 'fi‘d' ; 7177‘ {itnrJ‘vfil-f In 3 MHI' r‘n(mipd+€, ""‘M’L “dig” T are“ WM“ rim/(tr mt ,ML my,” me A {Vi/h filmfion 14‘; “If; dm-inr'" M Tf/f Ag figmjuyaw 71) HIE flfokfaltffie f “W’th welt; M‘Hq Web/{1 fo/w/rsm :3?“ 2’3“! ’maéw‘e Ma mg tetra/at a, .m MN“? 5’ oé/ErV-E 9-1 ‘7' a 4:! H r-CGJu-J. “(afaylpfiflcfcer @qIOAP" . 49‘6"] kt? cheat 4’;— —“ 6n . 4ft! MA: T'f/ F‘H’ Hat/saws was was 161dewa fiw "ce M," f with Jam/UL dbbf-(panmff '5’“ yap“ it ""al ()0 Wan! Viv—e . can [tn ;-1 c) What type of ELISA is this? “PW”? ‘fl 7(3 5"" ctr/4% d 7— d) After running the ELISA you prepare a standard curve. Would the curve look like Fig. A or 4‘24, Fig. 13? Briefly explain your choice. fir A 450 ugtml TSH ugtml TSH Ft‘jfijé-amvt/e the fi/Mun4’ of We” ,‘Acr—eauf-BI' Lyra}, £1,th anfl'j‘flt‘h’am ro-P TJ/HI filo/3 [7H ban/VJ 7% “fin-“'6 TIA/M; (eff .- «it flew; g..— WVQ’ :LE:+“‘E~L 7J7} iii/3' r firefight/9’3 {a A61 fan, Points: make (/0qu were”. c/Len 1745M. Exam 2 Section: 607’ Winter 2007 9) (8 pts) Indicate by number the position on the dipstick of the following components of a drug test for TCH before the test is performed. (Note: 3 is the position ofthe Test line and 4 is position ofthe Control line. The arrow indicates the direction of capillary flow.) a) monoclonal anti-TCH antibody conjugated with gold H Li goat anti-mouse I gG i TCH conjugated to bovine thyroglobuljn 1 2 3r (T) 4 (C) b) If the result of a TCH drug test was negative where would you See a band(s) and L113? The, L; and wry-Mal 9e flee/1 [A pofiyQ-pm ? 7/ loam/e Hue. mi; '- TCH AL; canjwyareat Mm 994‘ ammo 4,,- ‘i'o 4M TC” ELMLC 714%,?"613 .315 Hoar—{i Witt/eke. M TCH air—em}; 4' 9/1 fire wave In ampere, in £311 ,4, gym {Ii-6f I'VLA 314,9 f; Mamba $3“ 7!} m flfi-E‘é'DMnJ 4"“ fire/{47>n 10) (8 pts) You are studying a new species of sea urchins. You suspect that in this species 3%“ HR urchins the inactivation of MAPK is due to a phOSphatase present in the sperm, specific for P- MAPK, and that the inactivation of MAPK isLot related to the increase in cytoplasmic Ca2+ that follows fertilization. To test this idea you set-up an experiment similar to the one you did in lab using this new species of sea urchins. If it were true that MAPK depliosphorylation is due solely to a phOSphatase that-is present in sperm, what would you expect to see on Western blots of the following samples using the two antibodies noted? U5e + to indicate band observed and — to indicate band not observed. {1 pt for a correct answer; lf2 pt off for each wrong answer; no pts deducted if space left blank.) Western using anti- TMAPK antibody Western using anti— Sample PMAPK antibody fertilized eggs unfertilized eggs + seawater unfertilized eggs + A2318? unfertilized eggs + DMSO BIBC103 we? Section: 6—93 Name: Maillva (4W7— Exam 1 11) (16 pts) You want to test the properties of a new chemical, Compound Z, at 4 different concentrations (undiluted plus three serial dilutions). The testing protocol requires a 1:3 serial dilution of Compound Z using DMSO as the diluent. Compound Z was expensive to synthesize so you cannot waste material. Calculate how you will perform the Serial dilution so that the final volume in each tube will be exactly 30 pl. The first tube will have undiluted antibiotic stock. The next three tubes will have the serial dilutions. You may not discard material from any of the tubes, except Tube 4, to get the final volume to 30 pl. :3) Calculations for dilutions: ‘Fl‘ntl valeW-L Cli‘lvxlrw Adar“! '5 “M4”? J“ 3"" flan/4M {v . J. 64““ Jae-e 70W?” '0‘” “ 3”" 174,164 . . . re [‘6 ’ W‘s/(LI— lO 1)) Dilutlons (fill-1n allblanks): “ ‘ 4 7‘50”? 1:3wa " /\/\/\/\ Li 9 pl Comp’d Z l pl Tube 1 pl Tube 2 pl Tube 3 _Q_plDMSO 3703,11DMSO 376’ plDMSO 370p1Dmso Tube 1 Tube 2 Tube 3 Tube 4 Comp‘d Z /\ l 3&6 Ha“ Eire g c) You set-up an experiment to test the effect of Compound Z on sperm motility. Three dig/gtrw identical tubes for each of the following samples are prepared and kept on ice until all samples are ready. The tubes are then placed at 16 °C. After 5 min they are put back on ice and each sample is immediately examined under the microscope For sperm motility. After analyzing the results you realize that there were two samples that you should have 3 included as controls. What are the two samples and fly are they important? Sample 1: 50 pl sperm + 25 pl Compound Z from Tube 1 Sample 2: 50 pl sperm + 25 p] Compound Z from Tube 2 Sample 3: 50 pl sperm + 25 pl Compound Z from Tube 3 Sample 4: 50 pl sperm + 25 pl Compound Z from Tube 4 Samples:{30,419,$62.3,-ML + lfv‘fi/ We M w, t m . ~ . 1 f,. 0 0 ? c/xA’Ir/at e ,1 I. 417%” A foetal hmm “Sex/éfrf/weafi 177‘ (“f6 é/€.P7::/ MM“ 7)” eta/y 4A6 txmuea may #3 [MM/e thd’binfl jib tamper-B ny—e’zljétw‘l 7LLM+ I ...
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This note was uploaded on 09/04/2008 for the course BIBC 103 taught by Professor Coleman during the Spring '08 term at UCSD.

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BIBC 103 - exam 2 - toukdarian - “if I (Y UL.- ' balsa-ow...

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