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

清華大學生科 02級林佳緯

指導老師：

中研院分生所

Dr. 簡正鼎

Dr. 皮海薇

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.

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

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………..

Material and ProcessMaterial: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

Protein expression1.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

Blue 218

Magenta 126

Green 90

Violet 43.5

Orange 33.9

Red 17.4

Blue 7.6

(unit:kDa)

His His-h2 His(I) His-h2(I)

sup pe

His-p2 His-p1 His-p2(I) His-P1(I)

Pe sup

Gst-phyl protein induction(BL21)

Three factors may influence

Our experiment

1.DNA itself

2.The concentration of IPTG

3.Attention(Design positive and negative control)

Protein expression(DH5α )

Check the quality of DNA1.E.coli culture2.Miniprep. Of plasmid DNA3.DNA agarose gel (Cut by Xho )Ⅰ4.DNA sequencing (His-phylPREST A; His-phyl c2;Gstphyl)

Gst-phyl protein expression(DH5α)

Gst-p1 Gst-p2 Gst-v

sup sup sup

Gst-p1 Gst-p2 Gst-v

sup sup sup

Non-induction

Through Gst-beads

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)

Gv Gp(Ni) Gp Gv Gp(Ni) Gp

through beads

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 BL21

G v Gv(ni) Gp Gp(ni)

pellet DH5α

G v Gv(ni) Gp Gp(ni)

pellet BL21

-3 0 302 380 400

EcoRⅠEcoRⅠ

XhoⅠXhoⅠ

HindⅢ

-5 0 302 380 400

EcoRⅠ EcoRⅠ

XhoⅠXhoⅠ

HindⅢ

BamHⅠ

409

BamHⅠ

His-phyl

(3+0.9=3.9kb)

GST-phyl

(4.8+0.3=5.1kb)

Gst-phyl303~400 DNA cloning

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

His-phyl302 DNA cloning

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 Hind Ⅲ ； Bgl Ⅱ

digestion

9.BL21 heat-shock transformation

10.Protein expression

His-phyl302 and Gst-phyl303~400 protein expression

His-h2 ;

His-phyl302 no.1

Gst-phyl303~400 no.4;

Gst-vector

I

nI

H-h2 Hp G-p G-v

pellet