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Quick List | |
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Service Terms Confidentiality Delivery Request to the Customers Why SinoBio How to order Frequently Asked Questions | |
SinoBio Biotech's Polyclonal Antibody Custom Services are fast, economical and provide you the ultimate in design flexibility. You can provide your own immunogen (synthesized peptide or recombinant proteins) or work with our world-class recombinant protein manufacture team to construct an epitope-specific peptide to meet your immunogenic design criterion or synthesize the epitope under our help. Diverse purification method can be selected such as protein A or protein G affinity, immunogen affinity purification in addition to just the immunoserum. The purified antibodies can be used for WB; IHC; Elisa or further labeling.
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antigen |
Peptide | protein | Time | |
| Customer Provided | 10mg >Cys tagged >85% >0.4 mg/ml no organic solvents |
10mg >80% >0.4 mg/ml no organic solvents |
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| by SinoBio | 20mg Cys Tagged >85% |
5-10mg >85% |
2 weeks | |
| Procedure | 1.KLH Conjugation | 10 mg peptide |
- |
3 days |
| 2.Immunization | 2 SPF rabbits. | 8-10 weeks | ||
| 3.ELISA tests | until titer reaches 1:4,000. | |||
| 4.Antibody purification | Affinity with
Protein G Affinity Purification with Protein A Affinity Purification with Immunogen |
1 week | ||
| Shipment | Don't purification | 3ml pre-sera 60ml antisera |
10mg peptide
if synthesized by SinoBio ELISA TEST Result |
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| Protein G/A purified | 100-150mg antibody | |||
| Immunogen Affinity | 2-10mg antibody | |||
| Optional services |
1.additional rabbits, 2.protocol extension, 3.testing (WB, IHC, IP) and more. |
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1. Peptide synthesis by SinoBio
Custom FMOC Peptide synthesis up to 20 amino acids (20 mg). 10 mg of free peptide will be delivered to the customer together with the antibody.
A cysteine will be automatically added to the C-terminus or N-terminus of the peptide, and conjugated to KLH
Assistance on designing peptide with respect to protein homology, antigenicity, hydrophilicity, and synthetic suitability.
Peptide purity is >80%.
Time:2 weeks
2. Peptide Provided by customer
If the customer provide synthesized peptide by themselves, the
peptide should be more than 10mg, the purity should be at least 85% and with a
cysteine at the C or N terminal of the peptide. A full Q.C datasheet is required
with the purity and amounts. SinoBio Biotech will conserve the property to
determine if the qc data are reliable or not.
Antigen provided by the customer: 10 mg of
peptide or protein, the concentration should be >0.4 mg/ml and the buffer cannot
contain organic solvents.
2. Peptide conjugation to KLH
Conjugation of 10 mg peptide (using
Cysteine) to KLH.
Optional: peptide can also be conjugated to
BSA or HSA.
3. Immunization and anti-sera production in rabbit
Conjugated peptides or recombinant
proteins are used to immunize rabbits.
Ten-week protocol to produce antibodies in 2 SPF
rabbits.
ELISA tests until the titer reaches 1:4,000.
Western blot analysis when ELISA reaches target
titer.
3ml of pre-immune serum and 60ml of final immune
bleeds.
Optional: Additional rabbits are selected if
more antisera is required.
Protocol extension is optional if the titer reaches
1:4,000 in 10 weeks. Or, it's free for 4 weeks extension and 2 additional
immuno.
Time: 8-10 weeks
4. Antibody purification
Purification of the antibody by protein
G/A.
Antibodies are eluted using both pH11 and pH2.5
buffers and neutralized immediately followed by dialysis in PBS.
Time: 1 week
Additional Testing is optional such as WB, IHC, IP
* Customers will get progress reports when peptides
synthesized, begin of immuno and when titers reach 1:4000. All expectance will
be reported to and discussed with the customer immediately.
Confidentiality
Your confidential epitope to us
by mail will be covered. We will sign non-disclosure or confidentiality
agreements when necessary.
A general template for MTA and
CFA are available and you may change the terms according to your requirements
and discuss it with SinoBio.
Delivery
Materials delivered to customers
include the following:
1. 60ml antisera
OR 100-150mg protein G/A purified antibody
OR 2-10mg Immunogen purified antibody.
2. 3ml pre-sera.
3. 10mg synthesized peptide OR 2-5mg
purified recombinant protein when provided by SinoBio.
4.
Manufacture reports, including the major process, interim results, quality
control.
Delivery time:
The
typical delivery time is 8-13 weeks according to the complexity of the project.
Time for shipment is about 4 business days. This may vary depending on where the
customer located and accidentally, delayed by custom check.
The final products will be shipped via fedex. DHL is optional according to the
customer£¦#8217;s requirements.
Request to the Customers
1. Tell us the
antigen sequences or accession number in genebank through the E-mail. SinoBio's
specialist will help you analysis the epitope and discuss with you which should
be better. Or,
2. Tell us the epitope or peptide sequence
you want synthesized. Or,
3.
Antigens provided by the customer should be:
>10 mg of peptide
or protein
>the concentration
should be >0.4 mg/ml
>the buffer cannot
contain organic solvents.
SinoBio Biotech will conserve the property to determine if
the Q.C. data are reliable or not.
Correlative Service
Protein
Expression and Purification Services.
Peptide synthesis (by our partners)
Epitope analysis
(Free)
DNA Synthesis
Western
Blotting
ELISA
IP and CoIP
Why SinoBio
Professional:
SinoBio Biotech focus on protein research only, and the focus makes us more
professional. All of the group leaders are come from big biopharmaceutical
company with more than 10 years experience on protein production. The company is
well equipped with Bioengenering fermentor, AKATA, HPLC and we can provide China
GMP standard for your product. The fermentation scale is from liters to
500liters. Final production is scale from milligram to tens of gram.
Expertise: SinoBio£¦#8217;s protein experts have more than 300 different protein
purification experience, involved in NINE biologicals development, most of them
are in market and some of them in clinical trails. They also developed about 40
qualified cytokines and tens of biological enzymes.
Quality: Diverse quality control method available for your choice such as
amino acid sequencing, MS, MS/MS, LAL test, HPLC in addition to conventional SDS-PAGE
analysis.
Speed: Fast turnaround time (~10 weeks for a basic order)
Price: Competitive prices as low as 800$
Confidentiality: Strictly confidential customer's data. SinoBio will sign
non-disclosure or confidentiality agreements when needed.
How to
order
Please download the order form to specify your information, billing
information, and project information;
Please email the
completed order form to info@sinobio.net
Please follow these instructions closely to speed up
your ordering process.
1. SinoBio Biotech adopt a non-disclosure policy to all
of our customers. To protect your proprietary information please mail, email
(info@sinobio.net) or fax (+8621-5079-8029) your confidentiality agreement to us
for sign. If intended, this should be done before any information is released to
us. If you don't have your own NDA/confidentiality agreement, you can find ours
at here. Feel free to modify it to suit your specific needs.
2. Call us (+8621-5079-8029) or email us info@sinobio.net, your gene/protein
sequence and particular manipulations you wish us to perform. Particularly give
clear instructions on the following options: codon optimization vs. no
optimization; host organisms; single host vs. multiple host optimization. Total
amount of final protein you required, the qualities and QC terms you required.
Frequently Asked Questions: Polyclonal antibodies.
1) What is the chance that your customer-designed antibody can recognize native
protein? How to get a single band in Western Blot test with your antibody?
2) How long the titer will remain after last booster? How to reduce the background
and to have a better chance to see reaction with native protein?
3) How to make a good antibody?
4) How much antibody we will expect to get after affinity purification?
5) How much antigen do you need for a custom project?
6) Can you help in the preparation of antigen?
7)
How do you measure the antibody titer?
8)
How are SinoBio Biotech's antibodies supplied?
9)
Do you offer immunogenic peptide sequence analysis?
10) What kind of guarantee do you offer for your custom antibody production and
services?
11) What is immunoaffinity purification?
12) Do I have to cleave the tag from my recombinant protein before sending it to
you?
13) What is the smallest antigen that will produce an immune response?
14) Should I use a polyclonal antibody or a monoclonal antibody?
15) Do you provide epitope analysis? Do you provide peptide synthesis?
1) What is the chance that your customer-designed antibody can recognize native
protein? How to get a single band in Western Blot test with your antibody?
It's very unpredictable if a designed peptide antibody against native proteins.
Most customers designed at least two peptides to generate the antibodies against
the same protein. We guarantee the antibodies should be against the peptides.
So, please do the ELISA test with the peptide, if the titers of the antibodies
are very low against the peptides, the antibodies are bad. We should redo them
for free, after we confirmed your results by ELISA.
It's very hard to identify the specific bands with too much background in
Western blots. I would like to suggest you to test the antibodies with the
immunoprecipitation (IP). Don't worry about the background, as long as the
target bands coming out. The most backgrounds can be removed by the
affinity-purification of the crude antiserum, and the purified IgG can be used
in most immunodetecting experiments. So, higher concentration of the antiserum
is recommended in the IP, (1:25, 1:50 or 1:100).
The idea is that the target protein is firstly pulled down by the IgG in the
antiserum, and then the target proteins can be probed by the anti-tag antibody
or the antiserum itself. This experiment can remove most backgrounds. The
outline of the protocol listed below. If the protein is cloned from the
expression of the constructs, I would strongly recommend you to use the
monoclonal anti-tag antibody as the primary antibody to do the Western blot, the
results will be much better than the antiserum.
2) How long the titer will remain after last booster? How to reduce the
background and to have a better chance to see reaction with native protein?
Usually, the titers of the antibody will keep at higher level from day 10 for
6-8 weeks since the 4th immunization. The reboot on the animal should increase
the titers of the antiserum at this moment, however, it may be also increase the
backgrounds in some cases.
Actually, it's very unpredictable, if a designed peptide antibody is against the
native protein. The reason is that native protein may be quite different from
peptides in many ways like three dimesional folded structure, glycolysation and
surface lipids, which all could mask the epitope. Please bear mind, the immune
system is only response to the epitope, but not the sequence molecule only. So,
in our industry, we only guarantee the antibody against the peptide but not the
native protein. Besides, based on our experiences, some antibodies did never
work in immunohischemistry, even the antibodies had been confirmed really
against the native proteins with other immunodetecting methods. We know somebody
to solve this problem by the mix antigens immunization of the animals. For
example, with the mixture of two peptides, one is come from the N-terminal and
another one C-terminal in same protein. The affinity-purification of the crude
serum also improves the property of the antibody with relatively increasing the
concentration of specific IgGs.
Some other clues to consider:
We would like to suggest you to change some experimental conditions first,
including lower antibody concentration, lower incubating temperature (4oC or
14oC), higher detergent concentration, blocking the membrane with higher
concentration of BSA or blocking the membrane with d£¦#111nkey and horse serum, and
the second antibody from other companies or species.
You may try to affinity purify the antiserum by the column coupled with specific
peptides. In our experience, most customers were very please with their
affinity-purified antibodies, because the purified antibody could remove the
background effectively. However, few customers were not so lucky, especially for
the proteins came from plants or bacterial.
If your protein is from plants or bacterial, you may try the different eluting
fractions from affinity column. This method works well in some our customers.
Another option is to make polyclonal antibody from chicken, it will remove the
cross reactions between the mammalian.
We offer the affinity purification to our customers. This service includes
coupling of 10 mg of free peptide to the affinity-resin (5ml), purifying
50-100ml of the antiserum, providing around 5-10mg of purified antibody together
with the ELISA data on the every stage of the purification. The price is $500
for 50ml of antiserum purification, and $600 for 100ml of antiserum. Please let
me know, if you have additional questions.
Boosting immunization of rabbits will help to increase the antibody titer of
antisera. So that's why we provide the option to our clients. We can process the
re-boosting option for you right away if you are interested in. Please let me
know your decision at your earliest convenience.
3) How to make a good antibody?
To make a good antibody, some researchers try to generate the antibodies by
different sites on the protein sequence, and ideally select 3 sites, N-terminal,
C-terminal and the middle, to do the immunization. Believe or not, the different
site peptide will give you different results. So, it's not surprise for that
author used different antibodies to do the different experiments. Another option
is that you may make two peptides and mix them to immunize the same animal.
Actually, both human and rat sequences are good for the antibody production.
The antibody raised by one of both sequences will recognized both
peptides. Please bear mind, the immune system is only response to the epitope,
but not the sequence molecule only. Sometimes, the antibody may be only against
the peptide but not the native protein, that's because the peptide form a
different epitope after the folding. So, in our industry, we only guarantee the
antibody against the peptide but not the native protein.
The human sequence antibody works in human tissue, so it's for sure the antibody
against native protein. In most case, if the antibody is against human protein,
it will be also against same protein in other species. This is an advantage of
the polyclonal antibody. I had been used a polyclonal antibody from rat sequence
to do lots of experiments with human and mouse tissues.
4) How much antibody we will expect to get after affinity purification?
The yield of affinity-purification is very diversity in different rabbit and
is depending on the immune response of the individual animal to the antigen. We
got 5 to 10 mg of 96-99% purified IgG from 50ml antiserum in most case. The
ELISA range of the purified antibodies is 1.5-3.0 with 1:1k dilution from
1mg/ml, over 80% of antibodies is above 2.0 ELISA titers. The efficiency is
almost 100% for the conjugation of Biotin to IgG.
5) How much antigen do you need for a custom project?
A standard two rabbit schedule uses 1mg of antigen (peptide, protein, etc.),
but please provide 1.5-2mgs of protein are recommended, especially if you
require ELISA or extensions. For affinity purification, 2-3mg of lyophilized
peptide and 3-5mg of soluble protein are required. We prefer a concentration of
1mg/mL or greater.
If the antigen is a peptide, 10 mg will generally be sufficient. Less may be
needed if your peptide is highly purified. For affinity purification of an
antibody, we will need an additional 5 mg of peptide, preferably in dry form.
If the antigen is a protein, 2-5 mgs at a concentration of 0.5-1 mg/mL is
generally sufficient. Be sure to let us know which buffer you included and the
protein concentration. We recommend 3-5 mg at a concentration of 0.5 mg/ml or
greater. For affinity purification of an antibody, 5 mg of soluble protein is
required to prepare the immunoaffinity column.
This applies to both monoclonal and polyclonal antibody development.
6) Can you help in the preparation of antigen?
SinoBio Biotech offers a wide variety of custom services, including peptide
synthesis and conjugation, as well as gene cloning and protein
expression/purification using expression systems of either bacteria or
baculovirus.
7) How do you measure the antibody titer?
Antibody titer is determined by ELISA. In this method, we coat 96-well
plates with antigen and wash repeatedly to remove unbound antigen. When a
KLH-conjugated peptide is the immunogen, we use unconjugated free peptides as a
test antigen in ELISA. Serial dilutions of antibody are added to the
antigen-bound wells and washed repeatedly to remove unbound antibodies. A
labeled secondary antibody is then added and the number of bound primary
antibodies is determined by colorimetric assay.
8) How are SinoBio Biotech's antibodies supplied?
Purified antibodies are supplied in PBS with a low level of preservatives.
The composition of PBS is 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM KH2PO4 at
PH7.2. Anti-sera are supplied in original liquid form without preservatives.
Antibodies can be stored at -80oC for several years. Multiple freezing and
thawing can denature protein, therefore, aliquoting for single use is highly
recommended.
9) Do you offer immunogenic peptide sequence analysis?
Yes, and its FREE. Our scientists will help you select a sequence that has
potential to raise a good antibody response. Just email your full length protein
sequence(s) along with any supplemental information to abservices@prosci-inc.com
and we will have a response in 1 to 2 working days.
10)
What kind of guarantee do you offer for your custom antibody production and
services?
This is a great reason for taking advantage of our free antigen design
assistance. If you choose one of the sequences we recommend for peptide
synthesis, we will guarantee an immune response to the antigen by ELISA
titration.
11) What is immunoaffinity purification?
The peptide/protein is bound covalently to CNBr activated Sepharose 4B or SL
gel in a column. The antibodies within the polyclonal pool that are specific to
this antigen are allowed to bind. The unbound antibodies and other serum
proteins pass through the column, and the antigen bound antibodies are eluted.
The resulting purified antibody is highly specific. ProSci will provide you with
the serum, flow through, purified antibody (a yield from 0.1 mg to 0.5 mg per ml
of serum), re-usable column to the antigen of interest and ELISA results.
12) Do I have to cleave the tag from my recombinant protein before sending it to
you?
Please cleave the larger tags (GST, MBP, etc.) as they can interfere in
specific antibody production. Small tags such as His, Myc, and FLAG do not need
to be cleaved prior to antibody production. Usually GST (Glutathione
S-transferse) and MBP (maltos-binding protein) tags are immunogenic simply
because they are large proteins that contain many epitopes. It is preferable to
use antigen proteins without GST or MBP tags. However, other small tags, such as
His-tag, Myc-tag and FLAG-tags, do not interfere with specific antibody
production.
13) What is the smallest antigen that will produce an immune response?
Approximately 10-12kDa is necessary for an immune response. Anything smaller
needs to be coupled with a carrier protein, typically KLH, so that the immune
system can recognize it.
14) Should I use a polyclonal antibody or a monoclonal antibody?
This will depend upon the specific applications and requirements of your
experiments.
Polyclonal antibodies will recognize multiple epitopes of an antigen. They are
also more likely to maintain recognition of antigenic epitopes even when modest
changes in conformation or aggregation occur. Polyclonal antibodies are also
capable of recognizing different epitopes with different affinities. Because of
this ability, polyclonal antibodies have a broader range of potential
applications than monoclonal antibodies.
Monoclonal antibodies recognize only one epitope of the antigen and are highly
specific to that particular antigen. They generally yield much lower background
because of this specificity. Monoclonal antibodies also are highly homogenous
and allow for unlimited production with sustained specificity.
15) Do you provide epitope analysis? Do you provide peptide synthesis?
SinoBio Biotech can help the customer to analysis epitope for
better antibody production. And the services is free.
Till now SinoBio Biotech don't provide peptide synthesis
service. The customer can provide synthesized peptide to SinoBio for KLH
conjunction to produce antibodies. Or SinoBio Biotech can ask our collaborator
synthesized the peptides for you.
