Shakti BioResearch LLC
Contract Research
& Assay Development Services
1 Bradley Road, Unit 401
Woodbridge, CT 06525
Tel. 203-641-7827
www.shaktibioresearch.com [email protected]
Who We Are Founded in 2007
5,000 sft of laboratory space
Affiliated with GLP certified Laboratory providing Clinical
Research Service to Pharmaceutical Clients
Equipped with
Biological safety hoods
Incubators
Luminometer
Spectramax plate reader
BioTek ELx405 plate washer,
Thermal Cyclers
Other support instruments
Instruments calibrated regularly to maintain the consistency of
our work
Our Clients
Compound
Profiling
Assay
Development
Stable
Cell Line
Generation
Molecular
Biology
ELISA
Biomarker
Analysis
Protein
Production
Core Competencies
Pre-Clinical R&D
• SiRNA and gain-of-function studies
Target ID and Validation
• Cloning, Mutagenesis
• Stable cell line generation Molecular Biology
• Development of target specific biochemical & cell based assays
• In cell westerns, cell proliferation assay
• Compound profiling
Lead Optimization
• Bacterial, Insect and Mammalian Systems Protein Expression
Translational Medicine
Evaluate effects of kinase inhibitors on signaling pathways in cell lines
Evaluate surface marker antibodies for immuno-histochemicl analysis
Clinical Studies
Clinical PK analysis: Avastin, CRF
Biomarker analysis: VEGF, sVEGFR2, HGF and soluble cMET levels
Clinical Research
BIOMARKER ANALYSIS
ELISA
VEGF ELISA : Standard Curve
Spiked amt
pg/mL
QC
Samples Wells OD R
Conc
pg/mL
MeanConc
pg/mL Std.Dev. CV% Accuracy%
80 QC_Low C8 0.102 65.816 74.023 5.777 7.8 82.3
D8 0.115 76.852 96.1
E8 0.109 71.755 89.7
F8 0.117 78.552 98.2
G8 0.12 81.104 101.4
H8 0.107 70.057 87.6
600 QC_Mid C5 0.724 625.94 619.949 26.63 4.3 104.3
C6 0.69 593.538 98.9
C7 0.742 643.182 107.2
D5 0.688 591.639 98.6
D6 0.756 656.636 109.4
D7 0.706 608.759 101.5
1800 QC_High A5 1.815 1795.76 1864.926 114.397 6.1 99.8
A6 1.888 1884.247 104.7
A7 1.968 1982.895 110.2
B5 1.907 1907.515 106.0
B6 1.71 1670.975 92.8
B7 1.94 1948.166 108.2
VEGF ELISA : Method Validation
All QC samples within range
DEVELOPMENT OF CYTOBLOT ASSAY
Ce
ll L
ine
3
Ce
ll L
ine
1 / S
S
Ce
ll L
ine
4
Ce
ll L
ine
7
Ce
ll L
ine
6
Ce
ll L
ine
5
pAKT
250kD
150kD
100kD
75kD
50kD
37kD
25kD
20kD
15kD
Ce
ll L
ine
1
Ce
ll L
ine
2 / S
S
Ce
ll L
ine
2
Ce
ll L
ine
8 / S
S
Ce
ll L
ine
8
Screening Cell Lines For pAKT Expression
Cytoblot With S473 pAKT Antibodies
LY
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
Log Conc.(M)
%In
hib
itio
n
Wortmanin
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5
Log Conc.(M)
%In
hib
itio
n
Cytoblot With T308 pAKT Antibodies
LY
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-7.0 -6.5 -6.0 -5.5 -5.0 -4.5 -4.0
Log Conc.(M)
%In
hib
itio
n
Wortmanin
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5
Log Conc.(M)
%In
hib
itio
n
RECOMBINANT PROTEIN PRODUCTION
1. His-tagged protein
2. Thrombin treated His-tagged protein.
~2.75 mg protein digested overnight.
~90% protein got clipped
3. Purified protein. Biotinylated thrombin
removed by streptavidin agarose and
His-tag removed by Ni-NTA resin
Expression Of Recombinant Protein In Bacteria
1 2 3 MW
250kD 150kD
100kD
75kD
50kD
25kD
20kD
15kD
37kD
10kD
1 2 3 MW
250kD 150kD
100kD
75kD
50kD
25kD
20kD
15kD
37kD
10kD
4 - 12% SDS/PAGE
250kD
150kD
100kD
75kD
50kD
37kD
25kD
20kD
15kD
10kD
1 2 3 4
1. Control protein
2. Conditioned Media
3. Clone 65-18 Sup
4. Clone 65-18 Sup from
suspension culture
Precursor protein
Processed alpha subunit
Processed beta subunit
Expression Of Recombinant Protein In CHO Cells
COMPOUND PROFILING
Cell Titer Glo Assay (CTG)
Cell Titer 96 (MTS)
Parameter measured ATP MTS reduction
Incubation Time 10 minutes 1 - 4 hours
Sensitivity 50 cells 800 cells
Sample Type Suspension or adherent cells
Suspension or adherent cells
Detection Luminescence Colorimetric
Cell Proliferation Assays
CTG and MTS based
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-9.5 -9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5
Log Conc.(M)
%In
hib
itio
n
IC50= 19 nM
Cell Proliferation Assay CTG Assay
• Eight point dose-response curve in duplicates
• Compounds dosed for 72 hours
Luminescence Conc. (mM)
35756 40383 1
41029 44479 0.33
39864 40812 0.11
64838 66503 0.04
136117 139179 0.012
159310 166269 0.004
177348 168209 0.001
179366 173555 DMSO
Cell Proliferation Assay MTS Assay
• Eight point dose-response curve in duplicates
• Compounds dosed for 72 hours
• Absorbance read at 490 nm
-20.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
-8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5
Log Conc.(M)
%In
hib
itio
n
IC50= 342 nM
A490nm Conc (mM)
0.91 0.85 10
0.96 0.94 3.33
1.23 1.26 1.11
2.05 2.05 0.37
2.80 2.72 0.12
2.87 2.80 0.04
2.77 2.80 0.014
2.87 2.77 DMSO
Cell lines
Compound 1
IC50 (nM)
Compound 2
IC50 (nM) Mutation
HT29 20 21 BRAF
Colo205 20 17 BRAF
HepG2 17 15 N-Ras
HCT116 461 544 K-Ras
Caki1 > 1000 > 1000 WT
Proliferation Assay in Mutant Cell Lines
Mechanism of Action Assay Effect on ERK Signaling Pathway
Phospho - ERK1/2
Phospho - ERK1/2
Total - ERK1/2
Total - ERK1/2
Compound 1
Compound 2
DM
SO
0.0
37
m M
0.1
1 m
M
0.3
3 m
M
1.0
m M
STABLE CELL LINE GENERATION
TARGET VALIDATION
Ce
ll L
ine
1
Ce
ll L
ine
2
Ce
ll L
ine
3
Ce
ll L
ine
4
Ce
ll L
ine
5
Protein1
Protein 2
Protein 3
Protein Expression Profile In Cancer Cell Lines
b-actin
• Different cell lines were screened for the expression of proteins of interest
• Cell Line 1 and 4 expressed all three proteins of interest and hence chosen for siRNA studies
• Cell Line 1 had better transfectability
SiRNA Transfections In Cancer Cell Line 1
• Proliferation measured after 67 hours of transfection
• Two validated SiRNAs/ gene or four not-validated SiRNAs were used
• Gene A SiRNAs #5 and #6 had no effect on proliferation
• Gene B SiRNA #2 had significant effect on proliferation
• Gene C SiRNA #6 reduced proliferation by ~30%
• Gene D SiRNA #5 reduced proliferation by ~40%
0
50000
100000
150000
200000
250000
300000
350000
400000
No tr
eatm
ent
No R
NA
+ve -ve
Gen
eA 5
Gen
eA 6
Gen
eB 1
Gen
eB 2
Gen
eB 4
Gen
eB 5
Gen
eC 5
Gen
eC 6
Gen
eD 5
Gen
eD 6
Gene A Gene B Gene C Gene D
250kD
150kD
100kD
75kD
50kD
37kD
20kD
25kD
No T
rea
tme
nt
Mo
ck T
ran
sfe
ction
+ve
SiR
NA
co
ntr
ol
-ve
SiR
NA
co
ntr
ol
Ge
ne
D S
iRN
A #
5
Ge
ne
D S
iRN
A #
6
ProD
Actin
• Protein extracts were made 48
hrs post-transfection
• GeneD SiRNA #5 showed ~50%
inhibition in proliferation assay, and
reduced protein levels
• GeneD SiRNA #6 did not show
any inhibition in proliferation assay,
nor did it reduce protein levels
significantly
• Blot was reprobed with actin
antibodies (CST # 4967)
• Proliferation assay was for 72 hrs
Reduction Of Target
Protein In SiRNA
Transfected Cells