Production Description |
| Step 1) Phospho-peptides conjugated to KLH for immunization and to BSA for ELISA/non-phospho-peptide conjugated to BSA for ELISA |
| Step 2) Immunization of 6 Balb/c mice
for each peptide-KLH conjugate |
| Step 3) ELISA monitoring of anti-sera
development until titers reach >10,000 |
| Step 4) Fusion of spleen cells from 1
mouse of highest ELISA titer with SP2/0 |
| Step 5) 4-5 rounds of sub-cloning and ELISA screening to establish phospho-specific hybridomas |
| Step 6) Cryopreservation of cell lines |
| Step 7)Protein A/G affinity
purification of ascites from 2 cell lines |
Order Today To Follow This Production Schedule |
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Receive phospho-peptides/control peptides from customers;
Conjugation to KLH (for immunization) and BSA (for ELISA);
6 mice ready (take pre-bleeds)
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01-14-2009 |
9 |
Peptide Conjugation |
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1st Immunization (Complete Freud Adjuvant)
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2nd Immunization (Incomplete Freud Adjuvant)
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3rd Immunization (Incomplete Freud Adjuvant)
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After 3rd Immunization, IF titers>10,000, go on to Fusion and hybridoma cloning: as early as
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4th Immunization (Incomplete Freud Adjuvant)
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03-16-2009 |
70 |
Fusion |
SP2/0 fusion with spleen cells
from 1 mouse (plating in 10X 96-well plates)
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03-26-2009 |
80 |
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
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05-15-2009 |
130 |
05-29-2009 |
144 |
Cryopreservation |
Expand cell lines and make frozen
stocks
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06-19-2009 |
165 |
Ascites
Production/Purification |
Protein A/G purification of ascites
from 2 mice for each cell line
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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
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