T. Frey -- Biology 750 / Molecular Biophysics -- Assignment #2 -- March 18, 1998

Instructions: This is an open note open book take home assignment which is due in class Thursday, March 26, 1998! Work on your own, not in groups. Since you have plenty of time, there is no excuse for handing in a disorganized messy assignment. Copy your answers neatly showing all relevant calculations including all units and any graphs etc. required to determine the answers. If you use any materials other than your class notes or textbook, please provide complete references including page numbers. Also describe any help you receive from another person. The majority of credit will be given for the correct approach to a problem with less credit for calculating the correct numerical answer. If you have trouble working any part(s) of the problem(s) I will provide hints, but I may assess a penalty for any substantial hints I have to give to get you on the right track. In working the problems, try to obtain the most accurate answer you can, but you will have to make some simplifying assumptions in order to solve the problems. You must state clearly any assumptions you make and why they are necessary and/or justified. Finally, be careful of units and always use the appropriate units for each quantity in a calculation! Be as quantitative as you can in answering the problems.

Problem 1: How would you separate the four rRNA's described in Assignment #1 on a polyacrylamide gel by electrophoresis? Include in your answer a calculation of the appropriate gel concentration and where in the gel each rRNA would migrate to. Be as quantitative as you can.

Problem 2: You could also separate the rRNA's by gel filtration (a.k.a. molecular sieve) chromatography. Describe the characteristics of the column packing material you would use to accomplish this including a discussion of partition coefficients, void volumes, elution volumes, bed volumes etc. Sketch the elution profile from a column packed with the this material (you choose the column size) being as quantitative as you can. Clearly state whatever assumptions you must make about relevant parameters of your column packing material.

Problem 3: The data table below contains fluorescence anisotropy data for a fluorophore alone and for the fluorophore conjugated with a large protein. Analyze the data to determine:

(a) The rotational correlation times for both data sets

(b) Ao

(c) Does the data indicate whether the fluorphore when attached to the protein is free to rotate? Explain.

Note: You will find this question easier to answer if you can use a spreadsheet such as Excel. If you don't know how to use Excel but do know how you want to analyze the data, I will show you how to use the spreadsheet without assessing a penalty.

Fluorescence Anisotropy Data for Fluorophore

t (µs)

A

1

0.2195

2

0.1205

3

0.0661

4

0.0363

5

0.0199

6

0.0109

7

0.0060

8

0.0033

9

0.0018

10

0.0010

11

0.0005

12

0.0003

13

0.0002

14

0.0001

Fluorescence Anisotropy Data for Fluorophore-Protein conjugate

t (µs)

A

1

0.3767

2

0.3548

3

0.3341

4

0.3147

5

0.2963

6

0.2791

7

0.2628

8

0.2475

9

0.2331

10

0.2195

11

0.2067

12

0.1947

13

0.1834

14

0.1727

15

0.1626

16

0.1532

17

0.1442

18

0.1358

19

0.1279

20

0.1205

21

0.1135

22

0.1069

23

0.1006

24

0.0948

25

0.0893

26

0.0841

27

0.0792

28

0.0745

29

0.0702

30

0.0661

31

0.0623

32

0.0586

33

0.0552

34

0.0520

35

0.0490

36

0.0461

37

0.0434

38

0.0409

39

0.0385

40

0.0363

41

0.0342

42

0.0322

43

0.0303

44

0.0285

45

0.0269

46

0.0253

47

0.0238

48

0.0225

49

0.0211

50

0.0199