The amazing travel of researching for the Antibody of Protein “Phyllopod”

1. The amazing travel of researching for the Antibody of Protein “Phyllopod” 指導老師： 中研院分生所 Dr. 簡正鼎 Dr. 皮海薇 清華大學 生科02級 林佳緯

2. Phyllopod(400 a.a) Genetic analyses of phyl, sina and tramtrack(ttk) mutants indicates that phyl and sina function cooperatively to promote neural fate by antagonizing the activity of ttk. Further analyses show that the expression of phyl is negatively regulate by Notch signaling pathway. The phyl and sina were originally indentified to be required of the cell fate determination of phtoreceptor cell R7. Phyl is essential for the SOP transformation on notum.

3. The external sensory (es) organ development can be divided into several steps.

4. Original expectancy 1.Protein express well to gain enough “Phyllopod” protein 2.Inject the protein into rabbit, let the rabbit undergo immunoresponse to produce the antibody of “Phyllopod” protein 3………..

5. Material: His-phyl ; His-vector(pRSET A) Gst-phyl ; Gst-vector(pGEX-4T-1) BL21 heat shock E. coli DH5α electro-competent E. coli XL1-Blue electro-competent E. coli Process: His-phyl and Gst-phyl protein expression His-phyl302 and Gst-phyl303~400 DNA cloning Material and Process

6. Protein expression 1.Transformation(BL21 Heat-shock；DH5αelectroporation) 2.Culture O/N 3.Dilution for recoveration ； OD value 4.SDS-PAGE electrophoresis 5.Staining and destaining 6.Air-Dry His His-h2 His(I) His-h2(I) sup pe Blue 218 Magenta 126 Green 90 Violet 43.5 Orange 33.9 Red 17.4 Blue 7.6 (unit:kDa) His-p2 His-p1 His-p2(I) His-P1(I) Pe sup

7. Three factors may influence Our experiment 1.DNA itself 2.The concentration of IPTG 3.Attention(Design positive and negative control) Gst-phyl protein induction(BL21)

8. Gst-p1 Gst-p2 Gst-v sup sup sup Protein expression(DH5α ) Gst-phyl protein expression(DH5α) Gst-p1 Gst-p2 Gst-v sup sup sup Non-induction Check the quality of DNA 1.E.coli culture 2.Miniprep. Of plasmid DNA 3.DNA agarose gel (Cut by XhoⅠ) 4.DNA sequencing (His-phyl PREST A; His-phyl c2;Gst phyl)

9. Try:1.Low temp. induction 2.Lower density gel 3.Shorter fragment of DNA 4.Small-scale purification (Gst-beads) 10% Seperating gel His –p(pe) Gst-p(sup) Gst-v(sup) (I) (I) (I) Through Gst-beads Gv Gp(Ni) Gp Gv Gp(Ni) Gp through beads

10. Low temp. induction Low temp. O/N induction G v Gv(ni) Gp Gp(ni) pellet DH5α G v Gv(ni) Gp Gp(ni) pellet DH5α G v Gv(ni) Gp Gp(ni) pellet BL21 G v Gv(ni) Gp Gp(ni) pellet BL21

11. BamHⅠ EcoRⅠ HindⅢ EcoRⅠ -3 0 302 380 400 409 XhoⅠ XhoⅠ His-phyl (3+0.9=3.9kb) EcoRⅠ EcoRⅠ HindⅢ -5 0 302 380 400 BamHⅠ XhoⅠ XhoⅠ GST-phyl (4.8+0.3=5.1kb)

12. 1.Miniprep. Of Gst-phyl DNA 2.Digestion by EcoRⅠ 3.Agarose gel electrophoresis 4.Excise and extraction of DNA 5.Gst-phyl303~400 DNA Ligation 6.XL1 E.coli electrotrasformation 7.Miniprep.of .Gst-phyl303~400 DNA 8.Check by EcoRⅠ；BamH Ⅰ digestion 9.BL21 heat-shock transformation 10.Protein expression Gst-phyl303~400 DNA cloning

13. 1.Miniprep. Of His-phyl DNA 2.Digestion by EcoRⅠ 3.Agarose gel electrophoresis 4.Excise and extraction of DNA 5.His-phyl302 DNA Ligation 6.XL1 E.coli electrotrasformation 7.Miniprep.of .His-phyl302 DNA 8.Check by HindⅢ ；BglⅡ digestion 9.BL21 heat-shock transformation 10.Protein expression His-phyl302 DNA cloning

14. His-phyl302 and Gst-phyl303~400 protein expression His-h2 ; His-phyl302 no.1 Gst-phyl303~400 no.4; Gst-vector I H-h2 Hp G-p G-v pellet nI