1. Q: What are the advantages of polyclonal antibodies?
A: Polyclonal antibodies are capable of simultaneously recognizing antigens with multiple different epitopes, with the ability to tolerate minor changes in the antigen such as aggregation or slight degeneration. Detection of denatured protein polyclonal antibodies is an ideal choice.
2. Q: What is the specific concentration of polyclonal antibodies in serum?
A: Most commonly, the change in serum specific antibody concentration is 0.05-0.2 mg/ml (based on the internal evaluation of Agrisera through hundreds of affinity purifications). In special cases, better polyclonal sera can contain serum (or even more) of specific antibodies 1-3 mg/ml.
3. Q: What are the advantages of monoclonal antibodies?
A: Monoclonal antibodies recognize only one epitope, which can be a disadvantage in some cases, such as immunoprecipitation and when used in immunoaffinity columns. Monoclonal antibodies are very good as primary antibodies for detecting antigens in tissues. In theory they should have a lower background than polyclonal antibodies, but this is not always the case. Monoclonal antibodies are highly homogenous and the results are highly reproducible (if other experimental conditions can remain unchanged).
4. Q: What is important when choosing a peptide for the immunogen?
A: Usually the C-terminus or N-terminus of a protein is the most exposed part of the protein. In addition, in order to mimic the behavior of the protein, the synthesized peptide should have a structure and charge similar to the "cut out off" of the protein.
therefore:
----C-terminal polypeptides, then N-terminal acetylation modification
---- From the N-terminal polypeptide, then the C-end application of amidation modification
---- The peptides derived from the internal sequence need to be modified at both ends of the peptide. The following factors are also considered:
----Whether there are other proteins in the protein family of interest, cross-reactivity should be avoided.
Is the crystal structure (or homologous protein) of the protein known? This will help scientists studying peptides find the best peptides for antibody production.
---- The final application of the produced antibodies? Natural or denatured technology?
5. Q: What kind of antigen(s) can be used for injection?
A: Natural proteins, recombinant proteins, peptide carbohydrates or other microbial, bacterial, and viral extracts can be used for immunization. Small molecules of 5-10 kDa are required to induce sufficient immune responses. Biotoxic materials are prohibited for immunization. Vaccination.
important hint:
If the antibody acts on a denatured target protein (eg, immunoblotting, fixed tissue immunohistochemistry), denatured antigens are preferred (eg, gel blocks, inclusion body proteins).
If the target protein of the antibody used is a native protein (eg, immunoprecipitation), a non-denaturing antigen is preferred (the protein is dissolved in a solution containing no denaturant).
---- Not all anti-peptide antibodies recognize natural proteins, so careful selection of peptide sequences is critical.
The antibodies prepared by induction of recombinant proteins in bacterial expression systems do not, in some cases, recognize native proteins. The reason may be that the protein antigen expressed in the bacterial cells is erroneously folded.
There is no guarantee that it will be very successful in any immune response.
Related references:
"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.
6. Q: What is the amount of antigen needed for immunization?
A: It depends on the immunogenicity of the antigen. In a standard protocol (for rabbits, goats or hens) we used: about 500 μg peptide/animal/15 weeks old, about 400 μg protein/animal/15 weeks old. ?
If the association between the selected animal antigen and antibody is low, a smaller amount of antigen (less than 10 μg) at the time of immunization is also acceptable. We can also use our own immunization program. If the protein needs to be concentrated, please note that in some cases, the protein will partially adhere to the membrane in the concentration device and some protein will be lost.
7. Q: Why does the antigen-induced response produce only some IgM antibodies?
A: Due to the lack of T cell stimulation (Goding, 1993), highly conserved mammalian proteins tend to be very weak, mainly leading to the production of IgM antibodies, although there are exceptions. If it is a conservative mammalian antigen, species with far-reaching relationships such as hens are used.
8. Q: How should the antigen used for immunization be prepared?
A: The antigen should be dissolved in a buffer solution (Tris, MOPS, Hepes). Insoluble antigens (inclusion bodies) are as good as soluble ones. The ideal antigen concentration is 1 mg/ml, however lower concentrations are also acceptable.
You can also send us the SDS-PAGE gel containing the target protein, and put the gel in water to decolorize, because acetic acid can further denature the protein.
Avoid: Additives that are toxic to animals, such as protease inhibitors such as PMSF, sodium azide, and the like. Acceptable: low amounts of SDS, imidazole, urea, hydrazine, nonionic detergent, EDTA or EGTA and polyacrylamide. The final amount added will depend to a large extent on the sample volume/antigen concentration. Please check.
9. Q: Which species to choose for immunization?
A: The advantages of using IgG (rabbit, goat) and IgY (chicken) antibodies for the same antigen:
---- Independently confirm that the expected target protein is detected.
- Antibody libraries have different properties and can be complemented by different techniques (immunoblotting, immunoprecipitation, etc.).
---- Generally, species that have distant genetic relationships with antigens are selected for immunization (eg, hens are very suitable for producing mammalian proteins with conserved sequences). Find out more about IgY.
10. Q: Can I test pre-immune serum or yolk from some animals before I start the experiment?
A: At your request, Agrisera will send you several pre-immune serum or yolk samples before you start your experiment. You can choose the animal with the lowest background signal for testing.
11. Q: Are antibodies produced in rabbits better than those produced in chickens?
12. A: The process of producing antibodies is a combination of antigen, specific animal immune response and testing. For mammalian conserved target proteins, we recommend the use of antibodies produced in chickens.
Because they are more distantly related in evolution, and their immune system responds better to conserved mammalian proteins, and these conserved mammalian proteins may not be recognized by the immune system of goats or rabbits.
13. Q: I have customized an antibody. How do I know that customized antibodies recognize the correct protein?
A: First, confirm by indirect ELISA analysis. The ELISA plate is coated with protein or polypeptide of animal immune reaction, and the serum is added to the plate for incubation, and then the secondary antibody sends a signal. This is the first evidence that antibodies produced by immunized animals recognize a protein or peptide of interest. However, this result may not directly correspond to the level detected in natural tissue. ELISA plates coated with well-exposed polypeptides or proteins may not occur in natural tissue proteins that contain only a few cells. Therefore, testing involves collecting materials that are known or that we suspect correctly express the protein of interest. Also included are tests with deletion or overexpression mutant extracts as controls. The use of recombinant proteins as controls in immunoblotting may provide us with some information, but in some cases, the antibodies produced recognize only recombinant proteins and do not recognize native proteins. Another risk is that our membranes are exposed for too short a time to display only the signal of the recombinant protein. Considering the expression level of a particular tissue, following certain developmental patterns, some proteins are difficult to detect. All of these factors must be taken into account for the success of the test.
14. Q: If my antibody is successful in an immunolocalization assay, can the antibody be used in immunoblotting and other assays?
A: Again, it depends on which part of the protein the antibody recognizes and which part of the protein is exposed after fixation or after immunoblotting. If the polyclonal antibody is a synthetic peptide, it recognizes the antigen pool and if the antigen is not exposed after immobilization - the antibody will not recognize the antigen. In some cases, the addition of 6 - 8 M urea to the buffer contributes to protein exposure.
15. Q: How much serum or eggs are available?
A: Goat: 200 ml serum/month rabbit: 50 ml serum/month hen: 25 eggs/month
16. Q: How do I store antibodies?
A: Antibodies in serum are a very stable form of storage for antibodies. Serum at -20 ° C or -70 ° C can usually be stored for many years. In some special cases, anti-peptide antibodies are stored for a shorter period of time. It can be stored at 4 ° C for a short time. In some cases, the operation should be more careful. The first step is to freeze at -20 ° C, and then it is better to freeze at -70 ° C.
The total IgG fragment (after purification of the IgG protein by the protein G matrix), the general antibody is stable in this form. They can be stored at -20 ° C or -70 ° C for several years. For short-term use, sodium azide (0.02% final concentration) or other preservatives may be added.
The total IgY fragment (an antibody extracted and purified from the yolk by the IgY antibody), the purified IgY fragment is very stable at room temperature (although we do not recommend room temperature as a storage condition). IgY can be stored at 4 ° C under conditions of 0.02% sodium azide (note: sodium azide inhibits HRP activity) or 50 ug/ml gentamicin sulfate. To avoid IgY freezing or storing in dry ice, IgY can be stored directly at -20 °C.
References: "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.
The yolk antibody in the yolk needs to be stored at 4 ° C in the presence of 0.02% sodium azide (note: sodium azide inhibits HRP activity) or 50 ug/ml gentamicin sulfate. Egg yolk should not be stored frozen, otherwise it would make it difficult to purify the antibody, and it would be more difficult to purify the antibody in the yolk after 6 months of storage.
Affinity Purified Antibody:
The most vulnerable antibodies should be carefully considered for storage conditions and each antibody tested experimentally. Affinity-purified antibodies directed against different epitopes differ in their stability. Some will precipitate directly after purification and the activity will still be present. It is difficult to predict storage conditions in advance for a given antibody ---- there are some alternatives for testing:
-20 ° C or -80 ° C
- 4 ° C + azide preservative (0.02%) or thimerosal
-20 ° C + glycerin; final concentration of glycerol 10 or 50%
-20 ° C + BSA final concentration of 0.05 -0.5%
IgG
In mammals, polyclonal antibodies are precipitated after affinity purification and may be precipitated directly or refrigerated overnight. Some antigens can stimulate the production of a class of IgG, which is called cryoglobulin, meaning they will precipitate under cold conditions. However, heating to room temperature can solve this problem. After the antibody is dissolved, the precipitate can be removed by centrifugation.
IgY
Chicken antibodies will also precipitate after overnight storage or after a few weeks. Heating to room temperature often helps those precipitates dissolve. Otherwise, the IgY solution before use needs to be centrifuged before being used for precipitation.
Antibody solutions are stored without preservatives and are at risk of bacterial contamination, which is often the most common cause of protein inactivation.
General advice:
For larger volumes of affinity purified antibodies, filtered antibody samples and aliquots of liquid avoid multiple freeze-thaw cycles.
---- Store protein at a concentration of 0.5 - 1 mg / ml.
---- Check the stability of proteins under different storage conditions, especially IgM.
important hint:
Sodium azide will inhibit horseradish peroxidase activity and interfere with some coupling methods and bioassays. However, when IgY (containing 0.02% sodium azide) was prepared as a primary antibody diluted 1:2000 in an ELISA or WB experiment, the sodium azide content was not affected by the dilution due to dilution. Other reagents can also be selected in the antibody solution to prevent bacterial growth:
----0.01% thimerosal
----50 μg / ml gentamicin sulfate
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