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

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)

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

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

As with science, there are always problems… Dilution series cut off too abruptly: Saturated at 0.25 mg/ml, 0.05 mg/ml, and 0.025 mg/ml. No detection after this, however Recheck pH and recipe of buffer “ antibody concentrations

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 0.0025 mg/ml

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)