Production Description |
| Step 1) Phospho-peptide and non-phospho-peptide synthesis at AbMART |
| Step 2) Phospho-peptides conjugated to KLH for immunization and to BSA for ELISA/non-phospho-peptide conjugated 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 |
Order Today To Follow This Production Schedule |
|
|
|
|
|
|
|
|
01-20-2009 |
22 |
Peptide Synthesis |
Phospho-peptide/non-phospho-peptide synthesis based on sequences provided by customers;
Conjugation of phospho-peptide to KLH (for immunization) and (both peptides) to BSA (for ELISA);
6 mice ready (take pre-bleeds)
|
|
|
|
01-24-2009 |
26 |
Peptide Conjugation |
|
|
|
|
|
|
|
|
|
1st Immunization (Complete Freud Adjuvant)
|
|
|
|
2nd Immunization (Incomplete Freud Adjuvant)
|
|
|
|
3rd Immunization (Incomplete Freud Adjuvant)
|
|
|
|
|
After 3rd Immunization, IF titers>10,000, go on to Fusion and hybridoma cloning: as early as
|
|
|
|
|
|
4th Immunization (Incomplete Freud Adjuvant)
|
|
|
|
|
|
|
|
|
|
|
|
|
|
SP2/0 fusion with spleen cells
from 1 mouse (plating in 10X 96-well plates)
|
| |
|
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
|
| 05-15-2009 |
147 |
|
|
|
Expand cell lines and make frozen
stocks
|
|
|
Ascites
Production/Purification
|
Protein A/G purification of ascites
from 2 mice for each cell line
|
|
|
|
1) 2 phospho-specific hybridoma cell lines (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) ELISA data;
4) Cell line isotypes
5) 1 mg of phospho-peptide and non-phospho-peptide
|
