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Antibody Service FAQ (Polyclonal) 

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?

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.

Can I test the antisera in a Western Blot using the peptide antigen as a positive control?

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 1ug/ml) and the blocking peptide approximately 2 µg/ml. "

How much purified pAbs or antisera will I receive with a standard package? Can I get more antisera or purified antibodies?

It depends on the host species you chose. For rabbits, you can get 1~5mg purified pAb, 10~50mg purified pAb or 100~120ml antisera. 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.

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?

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.

Are the final antibodies purified from the antisera pooled together from the two animals? Can I have them purified separately?

Yes, the antisera will be pooled together for purification unless specified otherwise. You can 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?

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.

Why there is no affinity purification service for mouse pAb package?

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.

Do you charge any fee if you fail to deliver pAb at your guaranteed titer?

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.