User-supplied custom polyclonal antibody production is designed for generating antibodies
against an antigen such as peptide or protein that the client provides. We can generate
polyclonal antibodies in rabbits, chickens, goats, guinea pigs, or mice against
both soluble and non-soluble recombinant proteins, fusion proteins, ESTs (Expressed
Sequence Tags), peptides (conjugated or unconjugated), enzymes, heat-killed bacteria
and viruses, and cells. Live or toxic substances cannot be accepted.
- Small protein (<10 kDa), such as peptides, organic molecules
needs to be to be conjugated to a carrier protein to make it large enough to elicit
a good immune response in animals.
- If providing a fusion protein made using a large fusion partner
(such as GST or MBP), we recommend cleaving the fusion partner prior to submitting
it for immunization. Large fusion partners tend to be highly antigenic, and separate
purifications are needed on the resulting anti- serum to eliminate antibodies produced
against these molecules.
Antigen Quantity
We can perform a complete custom anti-protein polyclonal antibody production protocol
with the amounts of antigen indicated in the table below and project extensions
or immunochemistry services may require additional antigen. We can start a custom
anti-protein polyclonal antibody production project with 50% of the total amount
of antigen needed. Additional quantities can be sent during the project's progress.
|
Host
|
No. Host
(per project)
|
Species
|
Min. Antigen
(Per injection)
|
Bleed Volumes
|
|
|
|
|
Pre Immune
(per host)
|
Col.Sera
(per host)
|
Total Sera
(per host)
|
|
|
|
Rabbit
|
2
|
SPF New Zealand White
|
650 -1000 µg
|
2 ml
|
~20 ml
|
60-80 ml
|
|
Guinea Pig
|
2
|
VAF Dunkin Hartley Female
|
650 µg - 750 µg
|
1 ml
|
~ 2 ml
|
5-7 ml
|
|
Mouse
|
5
|
VAF BALB/cAnNHsd
|
400 µg
|
100 μl
|
~ 200 μl
|
~ 500 μl
|
|
Rat
|
5
|
VAF Long-Evans Female
|
600 µg
|
100 μl
|
~ 500 μl
|
~ 2 ml
|
|
Chicken
|
2
|
White Leghorn Chickens
|
650 - 1000 µg
|
1 egg
|
~ 10 eggs
|
10-20 eggs
|
|
Goat
|
2
|
Boer, Nubian, Alpine or ToggenbergCrossbred Goat,
|
2.5 - 3.5 mg
|
5 ml
|
~ 250 ml
|
~1000 ml
|
|
Sheep
|
2
|
Suffolk
|
2.5 - 3.5 mg
|
5 ml
|
~ 250 ml
|
~1000 ml
|
|
Horse
|
2
|
Young Adults/Either Sex
|
2.5 - 3.5 mg
|
2 ml
|
~ 250 ml
|
~1000 ml
|
|
Bovine
|
2
|
Young Adults/Either Sex
|
2.5 - 3.5 mg
|
2 ml
|
~ 250 ml
|
~1000 ml
|
|
Antigen Concentration
Bio-Synthesis can work with most protein concentrations as long as this information
is provided. 1 mg/ml is a good target range, but higher concentrations are welcome.
We consider .5 mg/ml a minimum concentration in most cases to ensure maximum protein
volumes in each immunization. Buffered NaCl solutions are preferred. Please dialyze
out any urea or imidazole.
Antigen samples submission
Antigens may be supplied in solution, lyophilized, as gel slice, inclusion bodies,
insoluble aggregates, GST/His fusions, protein conjugates, etc. if you are uncertain
about any of the antigen requirements for custom polyclonal antibody production,
contact us online.
Ready to Inject Antigens:
Protein extracts, purified proteins, peptide-protein conjugates, and proteins bound
to solid supports (e.g., beads). These molecules usually require no further modification
or processing. For lyophilized antigen, please indicate the total amount of antigen
contained, if there is a preferred storage temperature (4°C, -20°C, -80°C),
and if there are any specific reconstitution steps needed.
Antigens in Solution:
Antigen supplied in solution should be submitted at a concentration of 1mg/mL or
higher (please specify the concentration, total volume, and total amount). Precipitation
is not problematic for use in immunization for custom polyclonal antibody production.
Buffer components requirements:
- PBS, Tris, < 4M urea are preferred;
- SDS and citrate buffer are also acceptable
- Solution does NOT contain detergents, as these are harmful
and often toxic to the animals.
- Services such as ELISA and immuno-affinity purification the
antigen needs to be soluble, and for immuno-affinity purification the protein cannot
be in Tris.
Antigens From Gel Slices:
When submitting protein contained in a gel after electrophoretic separation, the
following guidelines should be considered:
Nitrocellulose blots are not recommended for immunization;
The gel should be stained with Coomassie Blue and de-stained and rinse with:
30% Methanol
5-10% Acetic Acid
Fill remaining 60-65% with dH2O
Excise the band of interest and estimate the amount of protein;
With gel bands in particular, it is important to maintain a higher concentration
of the protein (2-5 mg/mL) and then run into the gel. If it precipitates, use a
lower concentration. The gel is further diluted in 2-2.5 ml of saline and 2 mL of
adjuvant. 100 to 200 ug of protein in 500 uL of gel is the maximum we can inject
into rabbits.
Place the gel slice(s) in a conical tube with a damp gauze or paper strip to prevent
the gel from drying out;
Avoid freezing or homogenizing the gel strip so that we can aliquot appropriate
quantities of the protein for each immunization;
Do not ship the gel slice with excess water or buffer;
Ship the gel slice on cold packs by overnight carrier
NOTE: We cannot perform affinity purification or ELISA when protein
supplied in gel strip cut from an SDS PAGE gel.
Proteins in gel slices or on nitrocellulose membranes. These antigens require homogenization
prior to injection into the host animal and incur a small processing fee.