Frequently Asked Questions

Agrisera goods do not contain chemicals that are included in the United States Toxic Substances Control Act (TSCA). However, in some cases, US customs need a TSCA form to be filled in. In such cases, an empty form will be provided to you by the courier company.

As Agrisera products are not subject to TSCA, the certification should be a negative one ("I certify that all chemicals in this shipment are not subject to TSCA"). The information needed beyond the negative certification is the importer's name, phone number, email address and signature, as well as a short description of the products (as specified in the form).

The TSCA form must be filled in and submitted by the importer (customer). A TSCA form provided by the exporter (Agrisera) will not be accepted. More information can be found here.

HS codes, also known as customs tariff numbers, are used to classify products for customs. This information is provided by the exporter (Agrisera) in the customs declaration (only applicable for exports to countries outside of the EU). All Agrisera products fall under the HS code 38221900. Variants of this code may apply.

1. Go to Quick order under the tab Ordering at the top of the page
2. Fill in the product numbers and quanities you wish to check in the table
3. Click Update rows. The stock balance for each item will be stated

The European Union’s revised directive on payment services (PSD2) enforces new requirements to ensure that electronic payments are performed with multi-factor authentication, to increase the security of electronic payments for the buyer. This is something that all banks within the EU must abide by. Due to this, banks in the EU will not, and cannot, accept payments from credit cards that do not meet these security requirements.

Many banks outside of the EU have the necessary multi-factor authentication as the default for the credit cards they supply, while some do not. Generally, most banks can offer multi-factor authentication, and while not always the default, it can be activated if requested.

If you are experiencing problems with credit card payment on the Agrisera website, please ask your bank to change the settings of your credit card to support the newest multi-factor authentication (and payments to foreign banks), or, if not possible to do for your current card, provide you with a card that does.

• Longer product shelf-life
• No risk of antibody degradation in the unlikely event of a delayed shipment
• Environmentally-friendly shipping, with no need for dry ice (the production of which is expensive and energy-consuming)

Yes, to a certain extent it is possible to predict the outcome before making actual experiments. What is worth checking is the conservation level of the peptide or protein sequence used to elicit a given antibody, to the protein you are planning to detect. If a sequence is not provided on our website, please send us an inquiry about this, together with the sequence of the protein you are aiming to detect.

There can be different reasons behind it:

• You obtained another batch in the second purchase. Please, check it on the tube which contains a specific lot number. Each antibody batch is unique and can vary in its binding properties. 
• Another secondary antibodies was used.
• Another reagent was used.
• Another set of samples were used.

Usually the C or N-terminal of the protein is used, as those parts of the protein are exposed. Also, to mimic protein behavior, the synthesized peptide should have similar structure and charge as the protein it has been "cut out off". Therefore:

• Peptides derived from the C terminal should have N terminal modified by acetylation
• Peptides derived from the N terminal should have C terminal modified by amidation
• Peptides derived from an internal sequence should have both ends modified

The following points should also be considered:

• Are there any other proteins from the family of interest, where cross-reactivity should be avoided?
• Is the crystal structure of the protein (or homologous protein) known? This would be helpful for the peptide chemist in searching for the best peptide for antibody production.
• What is the final application of the produced antibodies? Native or denatured techniques?

Immunization can be done using native proteins, recombinant proteins, peptides, carbohydrates or other compounds of microbial, fungal or viral origin. Minimum molecular weight needed to induce sufficient immune response is 5-10 kDa. Biohazardous materials for immunizations are not accepted.

Important notes:

• If the antibodies are going to be used on the denatured target protein (example: Western blot, immunohistochemistry on fixed tissues), denatured forms of antigens are preferred (protein in inclusion bodies).
• If the antibodies are going to be used on native target proteins (example: immunoprecipitation), non-denatured forms of antigen are preferred (protein in solution free of denaturing agents).
• Not all peptide antibodies will recognize native protein, therefore a careful choice of peptide sequence is of crucial importance.
• Antibodies made against recombinant proteins expressed in bacteria can in some cases fail to recognize native protein. The reason for this might be incorrect folding of the protein antigen when expressed in bacterial cells.

No guarantees can be given in advance for a success of any immunization program.

Recommended references on the subject: "Monoclonal antibodies: principles and practice" by James W. Goding, 1996, ISBN 0-12-287023-9; Publisher: Academic Press. Using Antibodies: A Laboratory Manual, E. Harlow and D. Lane, 1999, ISBN: 0879695447; Publisher: Cold Spring Harbor Laboratory Press. Hjelm et al. (2012). Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes. PLOS ONE.

Advantages of using both IgG (rabbit, goat) and IgY (hen) antibodies developed against the same antigen:

• Independent confirmation that the expected target protein is detected.
• Antibody pools with distinct properties, complementing each other in different techniques (Western Blot, immunoprecipitation etc.), making double staining possible.
• A ground rule for immunization is to choose an animal that is genetically distant from the antigen source (e.g. hens are very suitable for production of antibodies against conserved mammalian proteins). For more information about IgY click here.

Antibodies present in serum
Serum is a very stable format for antibody storage. In -20°C or -70°C, serum can usually be stored for years. In some specific cases, the shelf-life can be shorter for anti-peptide antibodies. For very short periods of time, serum may be stored at + 4°C. In some cases, more careful freezing with a first step at -20°C, followed by -70°C is beneficial.

Total IgG fraction (IgG antibodies purified on Protein G matrix)
Generally, protein G purified antibodies are stable. They can be stored in -20°C or -70°C for years. For short-term storage, add some azide to a final concentration of 0.02 % (or another preservative).

Total IgY fraction (IgY antibodies purified by precipitation from egg yolk)
Purified IgY fractions are very stable, even at room temperature (although we do not recommend it as storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Avoid freezing and thawing of IgY, and storing it on dry ice. IgY antibodies can be stored at -20°C.

"The IgY preparations were stable over time. No loss of antigen recognition was observed after storage for 3 years at + 4°C". F. De Ceunick et al. Journal of Immunological Methods 252 (2001) 153-161.

Egg yolk
Antibodies in egg yolk should be stored at 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Egg yolk should NEVER be frozen as this will complicate purification of the antibodies. After 6 months of storage, purifying antibodies present in egg yolk might be somewhat difficult.

Affinity purified antibodies
Affinity purified antibodies are the most fragile. Caution should be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some will precipitate directly after the purification, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives to be tested:

• -20°C or -80°C
• +4°C with preservatives like azide (0.02%) or merthiolate
• -20°C with glycerol at a final concentration of 10 or 50%
• -20°C with BSA at final concentration of 0.05-0.5%

IgG
Mammalian, polyclonal antibodies can precipitate following affinity purification. This can occur directly after purification, or overnight during cold storage. Some antigens will stimulate the production of a class of IgG, called cryoglobulins, which precipitate at low temperatures. Heating the cryoglobulins up to room temperature can solve this problem. The antibody solutions can also be centrifuged to remove precipitates.

IgY
Chicken antibodies can also precipitate when stored in the cold, wither directly overnight, or after several weeks. Heating the IgY up to room temperature often helps to dissolve those precipitates. Otherwise, IgY solution can be centrifuged to remove precipitation prior to use. Antibody solutions stored without preservatives are at the risk of being contaminated by bacterial growth, which is one of the most common reasons for protein inactivation.

General recommendations:

• For larger volumes of affinity purified antibodies, filter-sterilize the antibody sample and aliquot the solution to avoid multiple freezing and thawing cycles.
• Ideal storage occurs at protein concentrations around 0.5-1 mg/ml.
• In cases of IgM antibody production, check protein stability in different storage conditions.

Important note: Sodium azide will inhibit horseradish peroxidase, as well as interfere with some coupling methods and biological assays. However, the amount present in IgY preparation (0.02 %) can be washed away in ELISA or Western Blot when IgY is used as primary antibody at dilutions of at least 1:2000.

Alternative agents for preventing bacterial growth in antibody solution:

• Thimerosal at 0.01%
• Gentamicin sulfate at 50 µg/ml