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BIBC 103 - exam 1 - toukdarian

BIBC 103 - exam 1 - toukdarian - C_02 BIBC103 W07 Section 1...

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Unformatted text preview: C_02 BIBC103 W07 Section: 1 Exam 1 5 pages —- Write legibly. FOR ALL CALCULATIONS: Show your work to get full credit. You can use the back of the page for an answer if necessary - but must indicate you are doing so. I) (6 pts) What equation describes the relationship between absorbance (A) and transmittance (T)? - A:wlogrw 01" 14:105‘71: b) If a sample of bromophenol blue has 30% transmittance at 591 nm, what is its absorbance (A)? '3) A3~Iogflojlglj AW 2) (9 pts) A 5X stock solution of Dilution Buffer contains 0.1 % bromophenol blue (BPB), 25 % glycerol, and 0.5 mM NaOH. - a) How would you prepare 2 mi of a 1X stock of Dilution Buffer? é E 7, C L/ re 1; 5 V .- OC ' a r ’37, XC i) —(lX)C2,mIJ [heath V. :— Li ml f-l’ock Adv/DWWJJHL ”704,0 K‘Q A , f b) What is the concentration of the three components in the 1X stock? I‘ I - a .— ,_.a:.=r2 LLf' BPB. .1 x0 .. alg J, .4 Dewar-1‘31 (an L )V.5 (stint-Ii Joan! ( l?mp)£,‘1flLJ—- I (z I 133:! Glycerol: 7.1”}, ; ”2.5": ' 1;, ”C21, and.) ”on! ‘3 ' 4—0.1 :(L114) loan! L '°°"“)("{"‘” JCLCZAU C1 -_- 1’5 [435‘ '0 NOH: ‘ UM” m P 1‘. a (01)" abim (5 )L‘l ILL 62 i) I. c) The MW of BPB is 670. What is the molarity of BPB in the 1X stock? 3 .O’Lola: «oz—g 1.0221. :ZI‘W'A’to'SMI [00:11 X 6107 {00 "1f )5 1.2?3/ 3) (10 pts) What is t]: definition of 116K for a buffer? + T‘ke {OK r— a ELL scar (T MAP/KB Le, H" JO 1;, 7‘» clfipW—Ipn ,vh‘ovx rival-61’? are efccat/ «Hots/fin] g: 12,1 ‘5 f-l’mlal-e. ‘ b) When selecting a buffer to use for a particular enzyme assay an important consideration is to select a buffer with a pK at /close to the desired pH of the assay. Why? . \ T14» { ff d'PA-Q/ +1? enfwrfi m PH 015 1£L~€ f‘ptwil'ran bgffi‘ “PM enzyme wit-l {+4. cao’lfchMi' writwwi— #94er 15/ ’6’ ‘1";1‘6'3” “a ”Wand/s! F” W” 114496;, EAEXW “id“‘fioiv 0k- ;W [PM tact/m he, in 2 Mil. [Tmymansegg' “6751/,” c) What is the H“ cogcentration in a buffei'L/at pH 5? Show )Zour work. ‘ _ n ”o Coot-dig IO-PH: £H+ . [Hi] ' 5 ' j 1 . pointszfl BIBC103 W07 Section: £09. Name:__ “f Exam 1 4) (15 pts) Two groups are given a solution of bromophenol blue (BPB) prepared in 0.01 M phosphate buffer pH 8.5 and told to determine the BPB concentration in the stock solution by reading the sample in the spectrophotometer. The students in Group A take 3 ul of the BPB stock and add it to 37 ul of 0.01 M phosphate buffer pH 8.5. They then take 5 ul of the diluted stock, add it to a tube with to 995 u! water, and then measure the A591 in the Spectrophotometer. The students in Group B take 5 ul of the BPB stock and add it to 995 pl of water. They then measure the A591 in the spectrophotometer. a) Before taking the absorbance reading of their samples, the students in each group set the absorbance of the Spectrophotometer they are using to zero using a blank. What is the correct blank to use for measuring the absorbence of the BPB solution for Group A? Explain your _ answer. (What should be in a blank?) HA {clonal/C 3' 5M! .OIMFhW/ohNJ—E éuhéér— lat-ff“? _,L ¢¢f / ‘ Infide, «H4 Han/r6 fA-WLU“ be hflVE/flhr‘nj In {4% Qflgnfiz; ¥ifi7 A%%n+f exec/0% Hue ram/ate, lever/Ly 1-1244! 763-3, ”’7 - b) The sample prepared by Group A gave an A591 = 0.273. If the molar extinction coefficient for BPB at A591 is 72,800 in 10 mM phosphate buffer pH 8.5, what is the concentration of the orginal BPB stock the students were given. Fill in the blank with your answer but show a_ll your calculations. Concentration of BPB stock: . 0’ M Pagan .274”; (mm); Clem) . C: 37,?f/rf056 M firm (a Cb (3M2) Cl {#LMafl, Cit/7"" View 1%: " (Smea- : (mm/amen) 61—: if X16" M c) The sample prepared by Group B gave an A591 = 2.170. Can they use this value to accurately determine the concentration of BPB stock? Why or why not? (Explain f do not do thecalculations.) NOJ'HAK} (ma/VA Jaw/Mia} @krmi‘rtg “/(«L QnCE/HVWH’DA 19-; 5/96/4301: Pacer/LR gasp; (”L/W 3 Aifci refit/14+ «commie/7 “fat/[0L6 411% (out. k'fltt Chat). 13;: C‘an. rm MnCZAfl/ifi‘vgifflzw/ fie 4:.ng iv ¢e+ vaoL'L at Afytl2abfarladncflke’flfmjpoin :46 Match “Wu {.6441 i“ Mutant/@713 calcwtajh'M-I. B1BC103 W07 Section: CO ’2’ N2 Exam 1 5) (20 pts) You have a mixture cont ' ' two‘prfitfirns, A 31131332?th want to separate using ion exchange chromato y. The pI of Protein A is 8.4. , a) What is the definition of pi? _ ‘ . rho {O I i3” +L‘Q’ {FD €LBOM~O For/\f' biker-6 M {cflgf’rn 4""), Viv V181" chat/yflu b) What property of the resin used for ion exchange chromatography determines the separation & properties of the resin? [1‘6 ren‘h'ffi? ”-911“ O'DLWVJ'P/ dW/flm‘ef {‘4‘}, fwd/3'74”" finnfifl 5‘9Aefi'p :7" [AMI A: Mffi‘l'i’L-‘e "3+ da‘lflfle (BL 6"" ér’V.‘ Felt-k‘vel)’ 554% fire-7%.}; f. Protein B is an enzyme. You don't know the pl of Protein B but you do have a colorimetric assay [5‘79“ ~ for measuring Protein B enzymatic activity so you set-up an experiment to estimate the pI of Protein B by binding the protein mixture to the cation exchange resin CM-Sepharose in - phosphate buffer at different pHs. The results are as follows: r. CM-Se-harose resin + buffer buffer no resin ' * -, means no activity detected in supernatant; +, means activity detected in supernatant e) Why do you have to set-up samples containing the Protein Mixture + buffer only (no resin) \ (bottom row on table)? TIM f if L/LFOtL- au’ 4 @mfw/ uni/’51 rod} one, 5 L‘flx ”3&1 Vat/z)» Late 075 M rte/124 +0 V3475} ”51% :54: Few/29 (1) Based on these results, between what pH values is the expected pI of Protein B? WI h 7PM “be/3'17 J41 feiwéo/t {OH {'5‘ ,.. pH i 9 e) Given only CM-Sepharose resin and phosphate buffers at pH 6, 7, 8, and 9, describe how you ‘ would separate the mixture of the two proteins such that you end up with one sample with only ‘< (330$ Protein A in solution and one sample with only Protein B in solution. If the separation is not =va -7 possible using only these reagents explain why. (Be Specific.) K ‘ j wettest mu m Mme/LA), of movie/ax 71> M 44‘ 5 [0H bw‘tc‘FeV’ g dwell 614d {fl CM-erfotLarVaf€ "eff/3- 7.46 ' Ferrite/y Charyggl Frankfm f wrW fkgm (25451;)g 7L Ire/fin 61M} I WOWQ kfimm/e fie ke/xn am; 20474“: H“ M+o PH lower— a”, T‘Ir/ Wad were fleece Least/f, fl/ovzal‘m ,‘m d’L‘B flke‘f/I‘SW/ Ahzzgfilflfwmyfiyéf . oboe" [OI f/ Jrfferéa‘f'df Afmfighlgfirn fuming Mn [73,14 £3 75% Vest/13A 4" (9'6 ILA 4’0‘2/ Few/Vi AJunéWam Wz'vtA PH, éfl/IQFX I 6 ”\ ).\ information provided then ° . _ (Vi=23’lmi 'jstoiLl/L: ‘3 (”‘9le 01; flb‘e'w/ r94 '/"Lrt.7£ a. BIBC103 W07 Section: 60 7’ Name: Exam 1 6) (20 pts) The following table lists the fractionation range for Sephadex Resins used for size exclusion chromatography: Se - hadex Resin 1 000 — 5 000 1,500 — 30,000 (3100 4,000 — 150 000 G150 5,000 — 400,000 a) What property of the beads used for size exclusion chromatography determines the fractlogigw ran/geoffethe 1%)}; ~€ ,0 L 7PM La—CAJ f ”Lek/MIMI' 13/7” fi/aoiWonmLMh ML aj€ ' b) What size molecules will elute first on a Sephadex G50 column? _ Moffiéng/f wf‘i’i/t A: 0,“, aver-e 7 a will ezwale 14/724 be J’N)‘ mpg {dfjgr sateen J’K’L MN (0 0% m r—f’fX/I, You run a sample containing the proteins BSA (66,000 Da) and Peroxidase (31,000 Da) and the colored molecules Blue Dextran (MW 2,000,000) and Phenol Red (MW 378) on a column packed with Sephadex GlOO resin. After loading the column you start collecting 1 m1 fractions. When you have finished running the column you determine which fraction contains each of the proteins/molecules in the sample. This is an idealized column so each protein/molecule elutes in only one fraction and none of the proteinsfmolecules interact with the resin. The results are as follows: BSA: present in fraction #15 6 6 00 -o Peroxidase: present in fraction #22 7/ o o O 1/0 Blue Dextran: present in fraction #10 2. mi VL Phenol Red: present in fraction #32 f}; c) What is V0 for this column? How did you determine this? If you can’t determine the V0 from the information provided then explain why. V0: 4 ml Va a. my, sac mag, sew/M The V0 if f‘l’mat/ 7‘: W Va 9% fine flex/Van fa P7517 filth/74. $1M. 4 fxm/Lefef (mi sfihaf— fl 5.7%?— m/w” 4,1924% 7H4; I, 17.9, - n f ”TB/'6 n , . . d) Whtal‘fs Vi forfius (glumn? how didoyou detenn’cine this? If you can’t determme the V1 from The (Bf-f 64(%h{ “/7 +M V'e_ 9"“! avH' firs 41m ;?i finecfivnf bra-Ara ervv/ real r‘f Freq, ,Lf a c) If you had included the protein Cytochrome C (12,000 Da) in your sample, where would it have run on the column? Explain. be 1Q ,-e, b A); I—r‘ Wowbd Lucy—Q, (lame-t ousting-9% phenv/ new 3 ”Lida” . ace/mam sew “‘6 3%“- +M Cafe/rm fix an). 11‘1“] hare/sheaf beémurf‘e4/7Lf I ‘6 waoints: ii if éQ‘l’Mtaeo/i )9le lies! 4,44 peroxi'daL/i, but-IL f-h'l/ Iii/£5133, "HAG F/AOh‘am‘tl'M/i 104436 of +09: fis/wM/J. 131130103 we? Section: 60 2/ Name Exam 1 7) (12 pts) A student prepared 15 ml crude extract from a rutabaga (aka yellow tumip). She then tried two different ranges for ammonium sulfate fractionation of the same rutabaga crude extract to see which would result in purification of the enzyme peroxidase. She usod 5 ml of the crude extract for each trial and had 5 m1 CE left over. Afier the last ammonium sulfate precipitation for each sample, the pellet was resuspended in buffer then dialyzed to remove the salt. After dialysis the student adjusted the volume of all three samples to 1.5 ml wi it'onal bufier. She then measured peroxidase activity for the three samples and so for the original crude extract following the same protocol you did in the lab. She also determined the protein concentration for the three samples. Her results are as follows: Sample Volume of Treatment Protein Volume ofsample Enzyme « Samle activit assa AAtmin ti‘L‘Z’ -_——__ W 40—70 % (Nasser 14 mam! . a) What is the colorimetric assay you used to measure peroxidase activity in the lab? Write out 6 the reaction inciuding a_11 relevant information. wkefi-e «logy-4,“ 53, ml- {0M1 Mr The, HEM in‘c, arm)! WM, W/‘e’l We W~/“-€~d “we I of #5 F +11%“- _ . tar-£6 ' Pe/OXtJd/fi 7L ’43 raga” [it #zaa—fflfifakfdfife + 2'” C ,9.” 11“, 11) Which treatrnentts) (D#1, D#2, both or neither) resulted in a purification of peroxidase rorn~le 46:27:. I— 9 the crude extract? How did you determine this? (THIS IS NOT ASKING WHICH IS THE BES TREATMENT— your answer should discuss both treatments). 3 m firearm a New,“ at mm, (Ly/wire, exit/Mt bean/e fir 501961396 WWW «we 5"“ Ham/t Me {fiche-"C 4,6796%)! 5? LN 65‘ Anangmy/f 54/)! Ilf 4ft A, {Molar-r fled/”Hid Lcfi‘uf-‘F’y 4414-4 #41 CE I #/ I Lgff PWnU-FRVPA fir- WWXI‘Ae‘LFfi 444 fif’l/‘e WVoXFt-Vlflxfe weaefmef- {feeds MW? W434 of,» We“ . In“. / by measuring the A230 or using the Bradford Assay. LN a) W'thj does the pH of the sample affect the Bradford Assay but not the A230? I N 57:44.1ng Alf/n, (“f a-Fyéoké ’é-E’zdwwkfi (“7‘ “Jae/- .Z“ “Elwe 6“ are 4%ij only want! harm flvaem 54% {OlLAJ'I‘Of‘ din-d. Oat/“f War ‘A Aye/ft (Lg/14,34‘un‘fjflwy0 jut/7L - b) Why do you read the absécrbaiice otflsamples (and protein standards) at A595 for the #- Bradford Assay? a; f 14:6,.6‘ ~55; Ag; FY diva”? $3 max fie? 7’44—9/ 4 39216 l'l E’l/me, G ciy {3 6g tat/MP8 «35$ foiwfi‘m '21 ‘ Qf é é.) (o l/ lam/f MJJA3‘M50( {if/67% “LG/OWL? dance é; points: (4..., ”(5.; the mien; mm g was Max lamb (thick/china Anti-Mm, Yuma/8r WWji’J—i, ...
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