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From Protein Sequences Provided by Customers To Western, IP, IHC and Protein Phosphorylation Western Hybridomas
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 Production Description
Step 1) Design and synthesis of 3 peptides at AbMART based on protein sequences provided by customers
Step 2) Peptides conjugated to KLH for immunization and to BSA for ELISA
Step 3) Immunization of 6 Balb/c mice for each peptide-KLH conjugate
Step 4) ELISA monitoring of anti-sera development until titers reach >10,000
Step 5) Fusion of spleen cells from 1 mouse of highest ELISA titer with SP2/0
Step 6) 4-5 rounds of sub-cloning and ELISA screening to establish phoshpho-specific hybridomas
Step 7) Cryopreservation of cell lines
Step 8) Protein A/G affinity purification of ascites production from 2 cell lines
Step 9) Generate and optimize antibody data in customer-designated application

 Order Today To Follow This Production Schedule
Date
Day
AbMART Track
Production Steps
01-06-2009
1
Order Date
 
01-08-2009
3
Peptide Design
Peptide design based on protein sequences from customers; Peptide synthesis;
Conjugation to KLH (for immunization) and BSA (for ELISA);
6 mice ready (take pre-bleeds)
01-26-2009
21
Peptide Synthesis
01-25-2009
20
Peptide Receive
01-26-2009
21
Peptide Conjugation
01-27-2009
22
Adjuvant Addition
01-28-2009
23
Production Start
01-28-2009
23
IMM-1
1st Immunization (Complete Freud Adjuvant)
02-11-2009
37
IMM-2
2nd Immunization (Incomplete Freud Adjuvant)
02-25-2009
51
IMM-3
3rd Immunization (Incomplete Freud Adjuvant)
03-07-2009
61
BLD-M1
Bleed; ELISA
After 3rd Immunization, IF titers>10,000, go on to Fusion and hybridoma cloning: as early as
Date
04-15-2009
03-11-2009
65
IMM-4
4th Immunization (Incomplete Freud Adjuvant)
03-21-2009
75
BLD-M2
Bleed; ELISA
Day
100
03-25-2009
79
BST-F2
Final Boost (iv/ip)
03-28-2009
82
Fusion
 SP2/0 fusion with spleen cells from 1 mouse (plating in 10X 96-well plates)
04-07-2009
92
S1-ELISA 1-Sclone
S2-ELISA 2-Sclone
S3-ELISA 3-Sclone
S4-ELISA 4-Sclone
S5-ELISA
4-5 rounds of sub-cloning and ELISA screening
05-27-2009
142
06-10-2009
156
Cryopreservation
Expand cell lines and make frozen stocks
07-01-2009
177
Ascites Production/Purification
Protein A/G purification of ascites from 2 mice for each cell line
07-15-2009
191
 Application Data Generate and optimize Western/IP/IHC/Phosphorylation data
07-21-2009
196
Shipment
1) 1 hybridoma cell line (Two 25 cm2 flasks with 2-5 x 106 cells in DMEM/high glucose/15% FBS);
2) 2-5 mg of Protein A/G purified mouse IgGs from ascites production;
3) Application data package;
3) Cell line isotypes;
4) 1 mg of synthetic peptide

2140 Xietu Road, Building 2 Shanghai, P.R. China 200032, Phone: (011)-86-21-64048593, Email: orders@ab-mart.com.cn