III EXPERIMENTAL RESULTS
INSTRUCTOR GUIDE Experiment 702. Analysis of a CellSurface Receptor
Part A: Slides 2 and 3
Slides 1 and 4
I CONTE NTS OF THE CHEMICAL PACKAGE Quantity Transfer pipets 1 0 10 x Con A Buffer - The working buffer contains
3
0
0.15M NaCI, 0.1mM MnSO 4 0.2% BSA and 10mM Tris-HCI, pH 6.8. Con, A-Peroxidage 0 . 8 0 . I m M - Dissolved in Con A buffer. C a C l Galactose (I M) - Dissolved in Con A buffer. 1 . 0 2 Mannose (1M) - Dissolved in Con A buffer. 1 . 0 , Hydrogen Peroxide 1 . 0 Tris Buffer (IM) 1 . 0 Chloronapthol 1 . 0 Con A - Dissolved in Con A buffer 0 . 5
ml
ml ml ml ml ml ml ml
II Preparation of Solutions Distilled or deionized water is required to prepare the solutions listed below. The water can be purchased from your local supermarket. All stock solutions except the chloronapthol should be warmed to room temperature before dilution.
Part B Slide #
2 3 4
% Erythrocytes in contact with other Erythrocytes 3-5% 40-60% 40-60% 3-5%
IV Answers to Study Questions 10 x Con A Buffer - To prepare the working buffer, add the 30m1 of 10 x buffer to 270mlof distilledor deionized water. Store the buffer in the refrigerator. Peroxidase Substrate Solution - Add the following to 40m1 of water 0.8m1 chloronapthol, 0.8m1 of 1M Tris buffer and 0.2ml of H peroxide). This solution must be prepared immediately before use. 2 0 2 ( h y d r o g e n
1. Ma n n o se - Mannose binds to Con A sites, thus preventing Con A from binding to its receptor (see Figure 2-3). 2. A single molecule of Con A contains many binding sites for receptor interaction. Thus, the lectin forms bridges between erythrocytes causing them to stick together to form clumps. 3. A f fi n i t y Chromatography: Couple Con A to an insoluble chromatographic matrix such as activated agarose. Pass the membrane extract
containing the Con A receptor over the column and wash the column to remove non-receptor proteins. Receptor may then be alluded from the column wit h mannose.
Š 1989 by John N. Anderson