Antibody Technical FAQ
- If we make the antibody against a peptide with just one phosphoresidue, is it likely to still recognize the protein when it's phosphorylated at both sites?
- Can I test the antisera in a Western Blot using the peptide antigen as a positive control?
- How much purified pAbs or antisera will I receive with a standard package? Can I get more antisera or purified antibodies?
- What is the difference between Protein A/G purified and antigen affinity purified antibodies? When I have a mAb or pAb, what kind of purification method should I choose?
- Are the final antibodies purified from the antisera pooled together from the two animals? Can I have them purified separately?
- If I do not order test bleed, do you normally do an ELISA on bleeds after the 2nd, 3rd, and 4th immunization? Or do you just do an ELISA after the last immunization?
- Why there is no affinity purification service for mouse pAb package?
- Do you charge any fee if you fail to deliver pAb at your guaranteed titer?
- Should I use Monoclonal Antibodies or Polyclonal Antibodies?
A: Both Polyclonal and Monoclonal antibodies have their own advantages which make them useful for different applications.
Large quantities of polyclonal antibody are relatively quick and inexpensive to produce compared to monoclonal antibodies. They are non-specific in that they are capable of recognizing multiple epitopes on any one antigen.
- Can help to increase the signal produced by the target protein as the antibody will bind to more than one epitope.
- More tolerant of minor changes in the antigen, e.g., polymorphism, heterogeneity of glycosylation, or slight denaturation, than monoclonal (homogenous) antibodies.
- Useful when the nature of the antigen is unknown.
- More robust detection due to multiple epitopes.
- Due to recognition of multiple epitopes, polyclonals can give better results in IP / ChIP.
- Inexpensive to produce and timeline is short.
- Prone to batch-to-batch 亚博的官网地址iability.
- Multiple epitopes make it important to check immunogen sequence for any cross-reactivity.
- Hybridomas provide an immortal cell line with the ability to produce unlimited quantities of highly specific antibodies. Compared to polyclonal antibodies, homogeneity of monoclonal antibodies is very high. If experimental conditions are kept constant, results from monoclonal antibodies will be highly reproducible between experiments.
- All batches will be identical and specific to just one epitope, which is a particular advantage when manufacturing procedures must be standardized e.g. clinical tests and therapeutic treatments.
- The high specificity of monoclonal antibodies decreases background noise and cross-reactivity, which helps provide reproducible results, and ensures efficiency in affinity purification.
- Specific antibody characteristics can be identified and selected. For example, sensitivity requirements and cross-reactivity levels can be specified and monoclonal antibodies screened to identify any cell lines exhibiting the required characteristics.
- Antibodies may be too specific (e.g. less likely to detect across a range of species).
- More vulnerable to the loss of epitope through chemical treatment of the antigen than polyclonal antibodies. This can be offset by pooling two or more monoclonal antibodies to the same antigen.
- Expensive to produce and timeline is long for hybridomas.
- I need ready-to-use monoclonal antibodies. Should I choose roller bottle culture or ascites production?
A: If you do not mind native mouse IgG (1-10%) mixed in with the antibody, ascites antibody production is a little faster than roller bottle cell culture.
- Does GenScript offer any guarantees for your customized monoclonal antibody service?
A: For qualified protein antigen, GenScript can guarantee two ELISA and WB positive clones against the immunogen. For qualified peptide antigen, GenScript can guarantee two positive clones with a positive ELISA result against the immunogen. For other conditions, GenScript will provide a guarantee according to the immune response after immunization.
- Do you offer endotoxin removal service after antibody production? What is your standard (EU/mg)?
A: Yes, GenScript provides this kind of service. EU criterion is ≤3 EU/mg.
- Do you provide cell storage with GenScript after the cell lines are delivered to me?
A: GenScript provides free cell line storage for 6 months from the date of final delivery of the project. After that time, customers who desire that their cells be stored for longer are charged $38 per year/vial. GenScript also can expand the cell line to set up a cell bank for interested customers. The price for this service is $25.
Antibody Service FAQ (Polyclonal)
A: Yes, a single phospho-peptide antibody may recognize the protein when it's phosphorylated at both sites. But a dual phospho-peptide antibody may also recognize the protein when it's phosphorylated at only one site. Thus, it is very difficult (even impossible) to develop an antibody which only recognizes the protein when it's phosphorylated at both sites and doesn't bind when only one site is phosphorylated.
A: The molecular weight (MW) of the peptide antigen (unconjugated) is too small to be applied in traditional Western Blot (WB) tests. To validate the specificity of the target band in WB, we suggest running an "antibody blocking" control, where you use the peptide antigen to block the primary antibody. The recommended dilution of the bleeds is 1:1000 (equal to 1µg/ml) and the blocking peptide approximately 2 µg/ml.
A: It depends on the host species you chose. For rabbits, you can get 1~5 mg purified pAb (SC1180), 10~50 mg purified pAb (SC1031) or 100~120 ml antisera (SC1030). You can check for other host species here. To get more antisera, you can choose an extended protocol which comes with an extra charge. For this option, you must supply more antigen.
A: In simple terms, the affinity purification isolates peptide specific antibodies while protein A/G (Protein A or Protein G) purification purifies the total immunoglobin (Ig) from the serum. Therefore, the protein A/G purified antibody contains both antigen-specific antibody and also endogenous Ig of the host animal, which may not be specific to the antigen.
Usually for mAb purification, since the antibody is generated by a hybridoma, which produces antibody from one B cell, the concentration of specific antibody is very high, so protein A/G purification will be fine. For pAb purification, since the collected antiserum contains about 10% specific antibody and about 90% total Ig from the serum, antigen affinity purification is recommended.
A: Yes, the antisera will be pooled together for purification unless specified otherwise. You can have them purified separately. The additional package is SC1525.
A: We usually do an ELISA test after the 2nd, 3rd and 4th immunization. If you need the report, please let the production team know as early as possible. The report we send to you will reflect a 10-data point titration, but we only run a 5-data point titration for an initial rough evaluation.
A: The amount of antisera from 5 mice (0.3-0.5 ml serum per mouse) is not enough for antigen affinity purification. Therefore, only protein A purification for mouse, or protein G purification for rat is provided. If enough antisera is collected (>10 ml) from more mice, antigen affinity purification can be performed.
A: For pAb services, we will restart the project free of charge until the guarantees are met. If we still cannot successfully deliver the antibody, we will not charge any setup fee, except in the case of complicated projects which list a setup fee in the proposal.
Antibody Service FAQ (Monoclonal)