Biochemistry I Fall Term, 2003

Ligand Binding: Problem Set#4,   1. Fab Binding to Cocaine

This page simulates the outcomes of equilibrium dialysis experiments. Background on the technique, including an animation, can be found in the Lecture 12 notes.
Background on immunoglobulin (IgG) structure and function can be found in the Lecture 11 notes and in Campbell, Chapter 21.5. Briefly, the IgG protein has two light chains and two heavy chains with a combined Mr = 140,000 Da. The IgG in this problem is a monoclonal antibody that binds specifically to the cocaine (COC) hapten.
The Fab portion of this antibody is used in the binding experiments. It has Mr = 47,500 Da.
You have a 10.0 mg/mL (10-6 g/mL) solution of the anti-COC Fab.
You can add any concentration of the COC ligand to the dialysis cell ([L]total).
a) Use the resulting values of [L]free to calculate n and n/[L];
b) Plot the data on a Scatchard plot;
c) Determine Kd and n, the binding stoichiometry;
d) Submit your data, calculations, and the graph that yields Kd and n.
1. Enter a value for [L]total in this box:
[L] added = mM (10-6 M)
2. For the above value of [L]total, Calculate [L]free
[L]free = mM (10-6 M)
    (Each calculated [L]free value has a small "experimental error" added to it.)

Use the steps described on Topic #2: Ligand Binding Calculations to solve this problem.
Hint: 1. Calculate the concentration of the Fab in mM units before starting your data collection. This will enable you to plot n vs. [L]free as you get the data.
2. For your submitted graph, ignore the data <10% and >90% saturation when drawing a line. These values will have more relative error and they are not necessary to determine an accurate slope and intercept on the Scatchard plot. Your data should include 3-4 points below and 3-4 points above half-saturation.

Print an Answer Form for submitting your solution (Class handout).

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