Protein Expression Screening Using Plant Tissue Prints On Ni

Friday, November 21, 2003

Description
The first print of the stem will contain a lot of chlorophyll but the second or third print should reveal the position of antigens. This method transfers soluble molecules of all sorts to the membrane. Different types of membranes cal also be used to immobilize them. Thus, you can screen for soluble proteins, glycoproteins, polysaccharides, glycolipids, nucleic acids, and enzyme activities. You can probe the membranes with reagents such as polyclonal and monoclonal antibodies, nucleic acid probes, enzyme substrates, labeled ligands, and labeled lectins.

Procedure
1. Sow Arabidopsis seed in 60-well seed trays (1 seed per well). Water the compost before sowing. Suspend the seed in water and transfer them individually by pipette. Cover the seed tray with a domes plastic cover and do not water again until germination.

2. Grow plants in a controlled environment room at 22C with a 16 hr day length at 50% relative humidity.

3. After 6 weeks, use the floral stems for tissue printing.

4. Prepare a nitrocellulose membrane by soaking it in 0.2 M CaCl2 and blotting dry (use a 1 cm wide strip for each antibody to be used).

5. Cut the floral stem from a plant between the first and second node. Press firmly onto the nitrocellulose for 2 sec, three times per strip. If making multiple strips for probing with different antibodies, cut a fresh face on the stem before printing the next strip.

6. Incubate the strips in blocking solution for 1 hr at room temperature with rocking to block all of the protein-binding sites on the nitrocellulose.

7. Replace the blocking buffer with a 20-fold dilution of rat hybridoma culture supernatant containing antibodies in blocking solution. Incubate for 2 hr at room temperature with rocking.

8. Remove the primary antibody solution and soak the strips in PBS for 30 min with rocking, changing the PBS every 10 min.

9. Remove the PBS and add a 2000-fold dilution of anti-rat IgG coupled to horseradish peroxidase in azide-free blocking buffer. Incubate for 1 hr at room temperature with rocking.

10. Remove the secondary antibody solution and soak the strips in PBS for 30 min with rocking, changing the PBS every 10 min.

11. Remove the PBS and add HRP substrate solution which will produce a blue color at the binding sites. The color should develop within 5 min.

12. Rise the strips extensively with ddH2O.

13. Dry the strips and examine them under a dissecting microscope.

Recipes
PBS pH 7.2
1.8 mM KH2PO4
4.3 mM Na2HPO4
2.7 mM KCl
137 mM NaCl

Blocking Solution 0.1% (w/v) Sodium Azide
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
10% (v/v) Sheep Serum

0.2 M CaCl2

HRP Substrate Make up immediately before use
30 l Hydrogen Peroxide
5 ml 3 g/ml 4-Chloro-1-Napthol in Methanol
25 ml ddH2O

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Protein Expression Screening Using Plant Tissue Prints On Ni