|
|
SERVICE |
DESCRIPTION |
TIME |
| 1
|
Peptide Synthesis |
-
Custom FMOC Peptide synthesis up to 18 amino acids (20-30 mg). Peptide with
more than 18 amino acids will be charged per rata. We can provide assistance on
designing peptide with respect to protein homology, antigenicity,
hydrophilicity, and synthetic suitability.
-
HPLC purification if peptide purity is < 90%
|
1.5 weeks |
| 2
|
Conjugation |
-
Conjugation of 4 mg peptide (using Cysteine) to KLH
-
Conjugation to IKERSATM (a unique 28 kDa recombinant protein
from Immunokontact, conjugation using Cysteine residue at the N-terminal of
peptide) for western blotting analysis and other functional studies.
|
0.5 weeks |
| 3
|
Immunization
and anti-sera
production |
-
Immunization of 3 mice with standard protocol. Sera from mice will be tested by
ELISA using immunogens. Once an acceptable titre is obtained, hybridoma fusion
will be done using splenocytes from mouse with the best titre
-
Antigen requirement: 2 mg of KLH conjugated peptide (for immunization) and 2 mg
of free peptide for screening. 2 mg of protein will be needed for purified
recombinant protein.
|
6-8 weeks |
| 4
|
Hybridoma
Fusion and
screening of
specific clones |
-
Supernatants from the growing hybridoma wells will be screened by ELISA and
western blot using recombinant proteins (if the immunogen is recombinant
protein). If the molecular weight and cell type distribution of target protein
is known, we will test the ELISA positive hybridoma clones on cell lysate
blots*. If needed several clones will be selected and send to customer's lab
for further characterization. Clones selected by the customer will be subcloned
and expanded for cryopreservation.
|
2-6 weeks |
| 4
|
Large-scale
culture |
-
A one-litre culture will be grown from one of the selected clones.
|
3-4 weeks |
| 5
|
Purification |
-
The secreted antibody will be purified by Protein G affinity chromatography and
send to the customer. Approximate yield may be 20-30 mg.
-
Antibodies are eluted using both pH11 and pH2.5 buffers and neutralized
immediately followed by dialysis in PBS
|
1 week |
| 6
|
Total |
|
15-21
weeks |
