Lab Report: GARP 2

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Lab ReportGARP 2 Stains-All studiesJAK-2EGFR

• 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 (1-ethyl-2-3-(1-ethyl-naphthol1,2-d
thiazoline-2-ylidine)-2-methylpropenyl ? 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 Stains-all while others
proteins are stained red or pink
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Stains all interaction with Ethylene Glycol

• a 575nm
• ß 535nm
• ß a 500-510nm
• S 470nm
• J 610-650nm

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.

A
B
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.
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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
a 575nm ß 535nm ß a 500-510nm S 470nm J
610-650nm

• 30 Ethylene Glycol
• Control
• BSA
• PGA
• Calmodulin

20 Ethylene Glycol 10 Glycerol 5.
Control 6. BSA 7. PGA 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)
a 575nm ß 535nm ß a 500-510nm S 470nm J
610-650nm

• Control
• BSA
• PGA
• Calmodulin

• 5. Control CaCl2
• 6. BSA CaCl2
• PGA CaCl2
• Calmodulin CaCl2

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GARP 2 plus addition of CaCl2
a 575nm ß 535nm ß a 500-510nm S 470nm J
610-650nm
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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,20glycerol,10D2
  O,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 10
62kD
Fig A C, 1UP, 2FT, 3WT1, 4WT2, 5 WT3, 6
WT4, 7 WT5 ,8Elu1, 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

Normal media 2.0X 106 cells/ml
Insect express media 1.5X 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

SourceFigure from Ivans 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
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Time course experiment of the C -Terminus EGFR in
Cos-1 cells by transient expression
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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 receptorJAK2 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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SourceTRENDS in Endocrinology Metabolism
Vol.12 No.6 August 2001
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JAK2 expression without induction.
M1 2
M1 2
M 1 2
M 1 2
2 1 M
Lane 1 and 2 are the same samples Lane 1 loaded
10 microliter Lane 2 loaded 20 microliter
Cellline obtained from Dr Ning Yang.
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JAK-2 expression after induction
120kD
Induction doesnt 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