Production Description |
| Step 1) Build expression plasmid based on cDNA clones provided by customers |
| Step 2) Express and purify tagged (His- or GST-) recombinant proteins |
| 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 hybridomas |
| Step 7) Cryopreservation of cell lines |
| Step 8) Protein A/G affinity purification of ascites from 2 cell lines |
Order Today To Follow This Production Schedule |
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01-09-2009 |
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Peptide Design |
Design gene cloning strategy for expression plasmid vector construction;
recombinant protein production and purification;
6 mice ready (Pre-bleed is done)
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02-13-2009 |
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Protein Production |
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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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04-18-2009 |
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SP2/0 fusion with spleen cells
from 1 mouse (plating in 10X 96-well plates)
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04-28-2009 |
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S1-ELISA
1-Sclone
S2-ELISA 2-Sclone
S3-ELISA 3-Sclone
S4-ELISA 4-Sclone
S5-ELISA
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4-5 rounds of sub-cloning and ELISA
screening
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06-17-2009 |
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07-01-2009 |
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Expand cell lines and make frozen
stocks
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07-22-2009 |
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Ascites
Production/Purification
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Protein A/G purification of ascites
from 2 mice for each cell line
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1) 2 hybridomas (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 synthetic peptide
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