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

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