1. integral spicule matrix proteins and BSA on Si and on calcite wafers, analyzed at SRC, Feb. 2011 The samples are labeled 48C, 72C, 48T, 72T, and BSA. All samples are at the concentration of 0.3 µg per ml. diluted by Yutao 1:3. he deposited 1 µl per droplet, and let it air dry. "48" and "72" indicate the stage of the embryos, "C" indicates the controls with the matrix proteins dialyzed against water straight from acid treatment “T” indicates samples neutralized with Tris after acid treatment and then dialyzed against water. what was the Tris pH? From Chris, the pH was 8. BSA is also in water.

2. Proteins on Si wafers: (2 or 3 droplets per wafer, coated with 1nm Pt) Ca movies: 48C and 48T gave type 1. 72C and 72T gave type 2 (true type 2?), however the peak 2 is small and SHIFTED. BSA gave no calcium signal at all. Ca spectra on Si.pxp Pupa thinks these spectra show Ca, not CaCO 3

3. Proteins on Si wafers: (2 or 3 droplets per wafer, coated with 1nm Pt) C movies: I scanned 48C and 72C only, they gave no 290.3eV spectra when the silts are 100µ and 150µ. In addition, the Ca concentration from the proteins is very low, thus the carbonate peak is not evident even if the peak exists. Pupa thinks the Ca spectra show Ca, not CaCO 3. is it true? check C spectra

4. Ca spectra on Calcite.pxp Proteins on calcite wafers (2 or 3 droplets per wafer, coated with 40nm+1nm Pt): Ca movies: 48C, 48T, 72C and 72T gave type2 spectra in Ca movies, no calcite, no type 1. BSA gave calcite spectra. possibly from the underlying calcite wafer?

5. ”C spectra on Calcite.pxp” Yutao’s analysis and conclusions Proteins on calcite wafers (2 or 3 droplets per wafer, coated with 40nm+1nm Pt): C movies: 48C did not give 290.3eV peak when the slits were 100µ and 150µ (Yutao will try to check this). The other three (48T, 72C and 72T) gave 290.3eV peak when the slits were 50µ and 150µ. In addition, BSA gave a small 290.3eV peak. C spectra on calcite.pxp

6. Spectra comparison (48h) Calcium spectra on Si wafer from 48C and 48T

7. Spectra comparison (48) Calcium spectra on calcite wafer from 48C and 48T

8. Spectra comparison (72) Calcium spectra on Si wafer from 72C and 72T

9. Spectra comparison (72) Calcium spectra on calcite wafer from 72C and 72T

10. Spectra comparison (48C) Calcium spectra from 48C on Si and calcite wafers

11. Spectra comparison Calcium spectra from 72C on Si and calcite wafers

12. 48C and 48T showed a weak Ca signal on Si wafer, but NOT a CaCO 3 (no peaks 2 and 4), then they gave type 2 spectra on calcite wafer. 72C and 72T on calcite wafer and Si wafer gave “type 2” spectra. However, the peak 2 is shifted.