Transcript Lab Report: GARP 2 & Stains-All studies JAK-2 EGFR

Lab Report:
GARP 2 & Stains-All studies
JAK-2
EGFR
Harpreet.K Dhiman
Department of Pharmacology
03/27/06
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Aim
 To investigate if Stains all dye could be used
to explore the conformations of GARP-protein.
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Stains all

Metachromatic cationic carbocyanine dye “Stains-all” (1ethyl-2-{3-(1-ethyl-naphthol[1,2-d]thiazoline-2-ylidine)-2methylpropenyl}
 It can bind to highly acidic proteins .

It can also be used to distinguish calcium-binding proteins
(CaBP) from others. CaBP are stained blue or purple by Stainsall while others proteins are stained red or pink
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Stains all interaction with Ethylene
Glycol
Stains all with Ethylene Glycol
1

Absorbance
0.8
β
3%
0.6

10%
30%
α
0.4
60%
98%
0.2
7.9M in Eth.Glycol


-0.2
673
642
612
581
550
520
489
459
428
400
0

α = 575nm
β = 535nm
β α = 500-510nm
S = 470nm
J = 610-650nm
Wavelength
All the further experiments were conducted in 30% ethylene glycol .
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Spectra of Stains-all + Calmodulin
Fig A: Shows the spectra of dye/protein ratio of 12.5. This was
performed with fresh Stains-all.
Stains all + Calm.
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A
B
2.5
Absorbance
2
Original
1.5
3x diluted
1
0.5
673
642
612
581
550
520
489
459
428
0
400

Wavelength
We checked different dye to protein ratios and concluded that
dye/protein12.5 is optimum for the induction of the band at 650nm
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Time course experiment to find the minimum time required for
interaction of protein with stains all, we looked at increase in
absorbance at 650nm , as this band is induced by the resulting
interaction.
Spectra of SA/Calmodulin (cicle every 10 min)
650nm - 700nm
0
10
1.6
20
30
50
1
60
70
0.8
80
100
0.6
110
0.4
120
0.2
150
0
170
Wavelength
673
642
612
581
550
520
489
459
428
160
180
190
Absorbance
40
1.2
400
Absorbance
1.4
0.25
0.23
0.21
0.19
0.17
0.15
0.13
0.11
0.09
0.07
0.05
0.03
0.01
-0.01
0 10 20 30 40 50 60 70 80 90 10 11 12 13 14 15 16 17 18 19
0 0 0 0 0 0 0 0 0 0
time in minutes
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

From our previous experiments, we conclude that dye/protein ratio of 12.5 is optimal
to induce the 650nm band.In order to make this experiment work for less
concentration of protein, different concentration of stains all was tried keeping the
ration of dye/protein 12.5
The experiment showed that 20μM dye is the minimum concentration, where we can
induce the 650nm band. For that we decided carry out all further experiments with a
dye concentration of 20μM.
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Interaction of Polyglutamic Acid (PGA);Bovine Serun
Albumin(BSA) and Calmodulin with Stains-all
Absorbance
1.4
1
1.2
2
1
3
0.8
4
5
0.6
6
0.4
7
0.2
8
673
642
612
581
550
520
489
459
428
400
0
α = 575nm
β = 535nm
β α= 500-510nm
S = 470nm
J = 610-650nm
Wavelength
30% Ethylene Glycol
20% Ethylene Glycol + 10% Glycerol
•
Control
5.
Control
•
BSA
6.
BSA
•
PGA
7.
PGA
•
Calmodulin
8.
Calmodulin
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Interaction of Polyglutamic Acid (PGA); Bovine Serun Albumin
(BSA) and Calmodulin with Stains-all plus addition of CaCl2 (1mM)
1.4
1
1.2
2
absorbance
1
3
0.8
4
0.6
5
6
0.4
7
0.2
α = 575nm
β = 535nm
β α= 500-510nm
S = 470nm
J = 610-650nm
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673
642
612
581
550
520
489
459
428
400
0
Wavelength
1.
Control
5.
Control + CaCl2
2.
BSA
6.
BSA + CaCl2
3.
PGA
7.
PGA + CaCl2
4.
Calmodulin
8.
Calmodulin + CaCl2
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GARP – 2 plus addition of CaCl2
0.6
α = 575nm
β = 535nm
β α= 500-510nm
S = 470nm
J = 610-650nm
Garp
0.5
1uM
0.4
2uM
0.3
3uM
0.2
4uM
0.1
5uM
694
1.4
1.2
1
0.8
0.6
0.4
0.2
Wavelength
673
642
612
581
550
520
489
459
428
0
400
absorbance
680
666
652
638
624
610
596
582
568
554
540
526
512
498
484
470
456
442
428
414
400
0
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Garp-2 purification for NMR
Aggregation is a big problem
 Buffers tried until now:

1)NMR Buffer exchange 20mM NaPi,20% glycerol,10% D2O
2) NMR Buffer exchange 20mM NaPi,20%glycerol,10%D2O,120mM salt
3)NMR Buffer exchange ( 20mM NaPi,20% glycerol,10% D2O, Increased salt 300mM & 500mM
Aggregation in all the buffers.
Then tried to elute protein in
presence of 5mM CaCl2
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Change in Elution Buffer.

100mM tris-Cl pH 8.0, 150mM NaCl, 1mM EDTA, 2.5 mM Desthiobiotin
100mM tris-Cl pH 8.0, 150mM NaCl, 1mM EDTA, 2.5 mM Desthiobiotin
NO elution of protein
1 2
3 4
5
6 7 8 9
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62kD
Fig A & C, 1=UP, 2=FT, 3=WT1, 4=WT2, 5= WT3, 6= WT4, 7= WT5 ,8=Elu1, 9= Elu2,10= Elu3,11=
Elu3, 12= Elu4, 13= Elu5, 14= Elu6.
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GARP-2 yield Comparison of the Insect
Express and the Serum supplemented
media



Cells were adapted to the insect express media in the spinner for 4 passages.
The cell count was found low for the cells in insect express media.
Cell count after 3days in spinner
Insect express media
Normal media
1.5X 106 cells/ml
2.0X 106 cells/ml
Cell concentration
was set same as 2.0X
106 cells/ml for both
the spinners
Spinners were
infected and
harvested
after 3 days
62kD
Conclusion: Bioexpress media leads to less production of the protein
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The transmembrane + juxtamembrane
Epidermal Growth Factor Receptor (EGFR).
Clone of C-term and Juxta+ c-term from Ivan
151
L1
312
CR1
481
L2
621
CR2
687
JM
955
Kinase
1186
CT
644
Extracellular portion
Intracellular portion
Source:Figure from Ivan’s presentation.
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Time course experiment of the C + Juxta membrane in cos-1 cells
by transient expression
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To find the location of the protein C-term+ Juxtamembrane EGFR
150 cm2 plate
harvested in 2
ml ofbuffer and
15 microliter
loaded in
corresponding
wells
60kD
60kD
25kD
Soluble protein obtained
by breaking the cells
.
Membrane solubilized protein in 4% OG , after break
opening the cells and separating the Soluble protein
obtained by breaking the cells
.
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Time course experiment of the C -Terminus EGFR in Cos-1 cells by
transient expression
24 48 72 96
72 96
150 cm2 plate harvested in 2 ml
ofbuffer and 15 microliter loaded
in corresponding wells
60kD
25kD
Soluble protein obtained by breaking the
cells.
Membrane solubilized protein in 4% OG , after break opening the cells
and separating the Soluble protein obtained by breaking the cells.
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Expression of the EGFR Bac-to
Bac expression system
Transfection is done
Need to find out the titer.
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JAK -2
•Signaling pathways activated by the growth hormone (GH)
receptor.
•An initiating event is probably the activation of JAK2 (Janus
kinase 2), a GH receptor-associated tyrosine kinase.
•Identification of the proteins recruited to the GH receptor–JAK2
complex and dissection of the signaling pathways that
aresubsequently activated will ultimately provide a basis for
understanding GHaction at the molecular level.
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Source:TRENDS in Endocrinology & Metabolism Vol.12 No.6 August 2001
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JAK2 expression without
induction.
M12
M12
Lane 1 and 2 are the same samples
Lane 1: loaded 10 microliter
Lane 2 loaded 20 microliter
Cellline obtained from Dr Ning Yang.
M1 2
Gel number
1
2
3
4
5
primary Ab
1:2000(1Hr)
1:3000(1Hr)
1:2000(1Hr)
1:3000(1Hr)
1:2000(0.5Hr)
M1 2
21M
secondary( anti Rabbit)
1:5000(0.5Hr)
1:5000(0.5Hr)
1:8000(0.5Hr)
1:8000(0.5Hr)
1:5000(0.5Hr)
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JAK-2 expression after
induction
120kD
Induction doesn’t really increase the yield of protein.
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Source STUART J. FRANK Etal : Endocrinology, Volume 135,No 5: pp 2228-2239
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Acknowledgements
Judith klein Seetharaman
Fernanda Balem
Hussien Baradia
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