Most frequently asked questions
What are the advantages of polyclonal antibodies?
Polyclonal antibodies are inexpensive and they have short preparation times (normally 4-6 weeks). Polyclonal antibodies recognize multiple epitopes of an antigen. They are more likely to maintain recognition of antigenic epitopes even when small changes in conformation or aggregation occur. Therefore they are a preferably choice for detection of denaturated proteins. 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.
Which animals are used to produce the polyclonal antibodies?
How many animals should be used?
Usually one animal is used for each antigen. Because the antigenic response is different from animal to animal, it is recommend that at least two animals for antibody production. Using more than one animal allows a wider response in terms of quality/affinity/specificity of antibodies. However, one animal might be enough if the antigen has been successfully used in the past to raise antibodies.
What type of antigens can be used?
SICGEN immunization protocols can be adapted to recombinant and native proteins, conjugated peptides and other haptens. Minimum molecular weight needed to induce sufficient immune response is from 10-12 kDa. In order to generate an immune response to small molecules, also known as haptens, conjugation to larger carrier proteins should be performed. SICGEN can conjugate these haptens to a carrier protein through the sulfo bond on amino acid cysteine to make the immunogen. For carrier proteins SICGEN often uses the large protein KLH (Keyhole Limpet Hemocyanin) to conjugate synthetic peptides. KLH compared to carrier proteins such as BSA (Bovine Serum Albumin) is useful since there is no reactivity with ELISA or Western Blots blocking reagents. Biohazardous materials for immunizations are not accepted.
In what form can the antigen be supplied?
Lyophilized, dissolved or precipitated in buffer (concentration 0.1-1mg/ml or higher), in nitrocellulose, in polyacrylamide gel – Coomassie-staining is possible; as pieces which are pH-neutral (as a result of being washed 2-3x in water) and kept moist (in distilled water), up to a maximum of 1000 µl of swollen gel per injection
What should be the purity of the antigens?
At leat 90-95% for proteins and 70-80% for peptides.
Do tags interfere with antibody production?
Usually tags such as GST (Glutathione S-transferse) and MBP (maltos-binding protein) are immunogenic simply because they are large proteins that contain many epitopes. It is preferable to use antigen proteins without these type of tags, however they can be used. However, other small tags, such as FLAG-tag, Myc-tag, V5-tag and His-tag do not interfere with specific antibody production.
What quantity of antigen is required per animal?
Protein solution – 1-2 mg at a concentration of 0.1-1 mg/mL is generally sufficient.
Protein in gel – 1 mg. Less amount might be sufficient.
Peptide – 10 mg is sufficient for all of our services. However, if your peptide is < 70% we need at least 7 mg; >70-80% we need 6-7 mg, if it is >90% we only need 4-5 mg. If you are interested in affinity purification of your antibody, we will need 3-6 mg of peptide, preferably in a dry form.
Conjugated peptide – 10 mg is generally sufficient. If the peptide is highly purified less amount might be sufficient.
Other Antigens – Please contact SICGEN.
Formailto:firstname.lastname@example.org affinity purification of the antibody, an additional 2-5 mg of soluble protein or 5 mg of peptide, preferably in dry form is required to prepare the immunoaffinity column. If other quantities or concentrations are available, please contact SICGEN. Provide information related to buffer and protein concentration used.
In the SuperFast and Standard protocols 500-600 µg of protein or peptide are used for the first immunization, 250-300 µg of protein or peptide for the other immunisations. In total 1,000 µg -1,200 µg (SuperFast) or 1,500 µg – 1,800 µg (Standard) of protein or peptide per animal.
Can the project be started without the total amount of protein or peptide?
Yes it can. Send to SICGEN the amount of immunogen available and the project will be started. The rest can be sent when it becomes available.
What information related to the antigen should be send?
Protein: molecular weight, purity buffer and concentration; recombinant protein: fusion moiety and expression system; native protein: source; Peptide: sequence, purity and quantity; conjugated peptide: sequence, protein carrier, purity and quantity.
(check on the order form)
How should the antigen be prepared for immunisation?
Proteins should be supplied in buffered saline solution (HEPES, phosphate, Tris-HCl, MOPS, etc.), as a lyophilizate or as a polyacrylamide gel slice. The slice gel should be destained in water to remove acids that can contribute to further denaturation of the protein. Desired antigen concentration is 0,1-1 mg/ml, however lower concentrations can also be accepted. Peptides should be in dry form unless it is conjugated with a carrier protein (such as KLH or BSA). When conjugated to carrier proteins the peptides should be sent in a buffered saline solution (no ionic detergents) with a concentration of at least 100 µg/mL. The following additives (maximum concentration) can be accepted in buffers if the concentration of the antigen is supplied at 500 µg/ml: 2 mM DTT (dithiothreitol), 1 mM 2-mercaptoethanol, 1 mM EDTA, 10% Glycerol, 200 mM Imidazole (in this case without NaCl), 0,1% Maltose, Manitol and Trehalose, 300 mM NaCl (in this case without Imidazole), 1 M Urea, 1 % Triton 100, 1 % Tween 20, 1 % b-octylglucoside, 0,1 % SDS.
Additives are toxic to animals, e.g. protease inhibitors such as 1 ug/ml Leupeptin, 0.1 mM PMSF, 0.1 mM DIPF, 1 ug/ml Pepstatin A, sodium azide should be avoided.
What can be done if the antigen is not soluble?
Usually it is possible to inject an insoluble antigen. If the particles can fit through an eighteen-gauge needle, it might work. It is recommended not to use detergents such as SDS to solubilise the antigen. The antigen / buffer mixture will be mixed to make an emulsion with the adjuvant to ensure a homogeneous preparation. You could also run multiple lanes of a gel and cut out the bands of interest and send us gel-imbedded protein as sample.
Does SICGEN offer peptide synthesis?
Yes, we use an outside source and it will take an additional 3-4 weeks. The average cost is 25€. per amino acid for 20 mg of <15 aa peptide at >70% purity (determined by HPLC/mass spectrometry).
What quantity of antigen is required for antigen affinity purification?
For purification of 50 ml antiserum, about 2-10 mg of protein or 3-10 mg of lyophilized peptide are required to couple to the affinity matrix column. The yield of antibody produced can range from 5 to 10 % of total immunoglobulins and is based on quality of immune response. Generally, a minimum of 10 mg of antibody can be obtained. If smaller amounts are available, less quantity can be used for purification. Please inquire SICGEN. The protein should not contain glycine or Tris buffers.
What is the yield using protein A/G affinity chromatography purification?
Most goat or sheep antiserum yields between 10 to 20 mg IgG per ml.
What is the yield by affinity chromatography purification?
Depending on titer anti-protein antibodies may produce 5 to 40 mg of specific IgG, while anti-peptide antiserum should yield 5-25 mg of specific IgG per 50 ml of antiserum depending on immunogenicity.
What quantities of antisera are as a rule supplied per animal?
Goat/sheep: 150-200 ml (SuperFast protocol) or 500-600 ml (Standard protocol). For extended imunisation protocols up to 200 ml serum/month.
What is the concentration of the specific, polyclonal antibodies in the serum?
Most commonly the concentration of specific antibodies in the serum is between 0.05-0.2 mg/ml of serum. In special cases, strong polyclonal serum can contain from 1-3 mg of specific antibodies per ml of serum (or even more).
In what form can antisera or antibodies be supplied?
Anti-sera are supplied in original liquid form without preservatives. Purified antibodies are supplied in PBS with a low level of preservatives (usually 0.1% azide). Antibodies can be stored at -80oC for several years. Multiple freezing and thawing can denature protein, therefore, aliquoting for single use is highly recommended.
How are the antisera tested?
The antisera are tested by ELISA as an internal quality control at day 28 (SuperFast) or at day 42 (Standard). Customized protocols varies. We recognize that quality and reliability are the most important to our customers, we never let an antiserum go out unless it has a minimum good titer (>20,000); when requested by the customer the extra ELISA assays for other bleeds will be charged according to the price list. It is also possible to test them by other methods, such as Western blotting and dot blot, with an extra charge.
What is the schedule of an immunization protocol?
SICGEN offers two types of immunisation protocols. The SuperFast and the Standard protocol (see the “immunisation protocols” page). However, it is possible to adapt them in accordance with the customer’s requirements regarding e.g. to the nature and the quantity of the antigen. Customized protocol is also availabe. Please contact SICGEN.
Is it possible to make any exception to the SuperFast and the Standard protocols?
Yes, it is. Uses ”Customized protocol” or please contact SICGEN.
Can an immunisation protocol that have been successfully used in the past be repeated for the same antigen?
Yes, it can. The protocol should be submitted on-line via ”Antibody Customized Protocol” to SICGEN for reviewing and commenting and then it will be followed. Please contact SICGEN for any specific requirements that do not fit into the standardized or customized protocols.
How long can the animals be kept?
There is no automatic final bleeding or disposal. It is possible to keep animals for a relatively long period of several months or years. The customer decides how long the animals are to be kept and when they are to be final bleeded or disposed of. On every occasion the antisera are supplied, the customer is given an email reply for communicating his further requirements with regard to animal maintenance.
Can the pre immune serum from few animals be tested before to start the project?
Yes, on request it will send few samples of pre immune serum before starting the project. The customer can test and choose most suitable animal with the lowest background signal.
How to send the antigen?
The best form is soluble or slightly insoluble. The total amount of antigen should be send in several 2 ml eppendorf tubes containing 0.250 mg each of antigen in PBS or other buffer. It is not necessary to send all the antigen at the start of the project; more can be sent later.
In Portugal: we recommend Post Office service for over night delivery (Correio Azul) or an express carrier (blue and dry ice packages).
In Europe/USA/Canada or rest of the world: please, use a reliable carrier.
Should the antigen be sent on dry ice?
The antigen should be sent at the conditions that the customer feel that the antigen will remain most stable. Usually, most proteins should be shipped on dry ice. Peptides and conjugated peptides in solution can also be sent on dry ice. Most lyophilized antigens and antigens bound to nitrocellulose membranes are stable at room temperature and can be sent at room temperature. Gel slices should be sent on ice packs (blue ice).
Can I use my own shipping company?
Yes, please provide us with the name and account number of your courier.
Which days does SICGEN ship the serum packages?
Shipments within Portugal and Europe are on Monday, Tuesday and Wednesday. To USA Canada and rest of the world customers only on Monday. We use overnight priority on all our shipments.
How much do SICGEN charge?
We do have fixed charges for antibody production projects (see immunisation packages). The on-line price list shows how much SICGEN charges for immunization packages, shipping or optional services. Using this system allows SICGEN to charge only for services that you require for your antibody project. Price estimates/quotations are available upon request, or use our automated Order or Quotation Builder.
What method of payment is accepted?
Payments can be made by credit card or by bank transfer using IBAN. Please note that any bank or service charges will be charged back to the customer. The bank account details for payments via Bank Transfer will be send with the invoice or can be sent by fax upon request by email. Credit card payment is also available.
What does the guarantee offer include?
The guarantee offer covers the following services:
– immunising one goat in accordance with SuperFast or Standard protocol
– titre testing by ELISA, with guaranteed minimum titres of 1:20,000 for a protein and peptide produced by SICGEN.
– supplying preimmune serum and at least 150-200 ml (SuperFast) or 500-600 ml (Standard) of antiserum having the guaranteed titre and the extra serum (500-1,000 ml) from the exsanguination.
– In order to select the most suitable animals, which do not show any reaction to the antigen in the serum and upon request by the customer, the preimmune serum from several animals can be tested by ELISA. This service will be charged as an extra.
What is guaranteed provided at the end of protein expression service?
You will get whatever amount of protein purified from 4 liters of culture with our best-optimized procedure. But neither yield nor purity will be guaranteed. If you get less than 1 mg purified protein and/or less 75% purity for antigens designed by SICGEN, no payment will be requested.