1. Protein Microarrays: Spotting and Calibration
Kyle Nordquist
Oral Presentation: 1/29/04

2. Where we left off…
Microarrays = method to simultaneously test mass quantities of molecular interactions
Purification of GST-tagged yeast proteins
Spotting onto specifically coated glass microscope slide (Glutathione)

3. Purification successful!
GST protein purification has been established, shown through WB.
Protocol optimized by myself and Joseph Tasto.
Purifying large enough amount?
181 kD
27 kD

4. Dilution Series: Concentration Determination
Take GST of known concentrations and make dilution series
Spot onto chip
Analyze using GenePix

5. As with science, there are always problems…
Dilution series cut off too abruptly:
Saturated at 0.25 mg/ml, 0.05 mg/ml, and mg/ml.
No detection after this, however
Recheck pH and recipe of buffer
“ antibody concentrations

6. Quick redo by hand…still have problems!
0.2 mg/ml
New dilution series made
Hand spotted (via micropipetter) onto slide
Fluorescently probed as before
Antibody concentration too high—all appear saturated.
0.1 mg/ml
0.05 mg/ml
0.025 mg/ml
0.01 mg/ml
0.005 mg/ml
mg/ml

7. Next Steps Redo hand spotting with correct antibody concentrations
Calculate concentration curve if above works
Purify more proteins (Dr. Link has decided to purify all the yeast translational control proteins for spotting, about 364 in all)