Custom Services

Concept, Programs & Schedules

Is there a standard project schedule?

We have best experiences when immunizations and bleeds are all about two weeks apart. Therefore, our standard schedule is tailored accordingly. However, if you have a very urgent project, a ten day distance between injections and between the last boost and the first test bleed can be an acceptable alternative. On the other hand, if speed is not a crucial factor, you may want to benefit from natural antibody maturation over time with four week intervals. This positive effect on antibody affinity is most pronounced with peptide antibodies.

As with any antibody program, we are flexible and will be pleased to adjust the schedule to your requirements.

In order to proceed with the large bleed(s), we need your feedback (based on your test results). Nothing happens without your consent. This is also true for the final bleed (exsanguination).

standard project schedule

Last update on 10.08.2011 by Matthias Reinhard.

"Parking a rabbit" - what is that option good for?

Instead of taking the final bleed (exsanguination), you might want to pause "park" the animal instead, in order to resume the program later with two additional boosts and 3[-4] large bleeds. We offer this "20 week elongation block" with particularly favorable rates. Please see our options (available with any antibody program) or ask us for details.


Major advantages of having the program continued for such a long time:

  • natural affinity maturation of antibodies
  • more economic than starting from scratch with a new rabbit

"20 week elongation block" (yielding an additional >45 ml of serum):

  • 10-12 weeks of pausing
  • 2 additional boosts
  • 3 additional large bleeds

Last update on 18.08.2011 by Matthias Reinhard.

Shall I use a recombinant antigen or a peptide for immunization?

Please make sure to read our special article about that critical question. If that doesn't answer your questions or if you have more specific questions please don't hesitate to contact us.

Last update on 09.08.2011 by Matthias Reinhard.

Should I use a mixture of two or even three peptides for immunization?

To minimize the risk that an anti-peptide antibody does not recognize the natural protein (~30%, average number from various sources/suppliers), some antibody service providers recommend to use a peptide mixture. Theoretically this yields an improvement according to the following equation:

Chance that at least one peptide will be successful = 100%-(30%)2 = 91%. This is a popular but naive fallacy: Antibodies that react with one of the peptides (chance: ~100%) but do not react with the corresponding protein are not simply "dead" but often recognize a wrong protein that contains an identical or similar sequence motif. Therefore, they will spoil the whole serum. That is why we strongly discourage such a practice, except for the case when individual peptide specific antibodies will be (fractionally) purified on as many separate peptide matrices as there are peptides in the mixture.

Actually, we do offer such a service, even with three different peptides - of course with fractional affinity purification on three distinct peptide columns!


For single peptide/protein antigens we guarantee that the immunization will result in antibodies recognizing this peptide/protein, mostly even with high titres (see FAQ on that topic). When two or more antigens are injected simultaneously, one or two of these might become the dominant antigen, actively out-competing or suppressing the response to the other(s).

For more details about this multiplexing immunization please see the article by Karin Larsson et al. ("Multiplexed PrEST immunization for high-throughput affinity proteomics" Journal of Immunological Methods 315 [2006] 110–120).

Therefore, antibodies against a given peptide maybe underrepresented in a setting with two or more peptides in parallel. Also, it is likely that the yield of total antibody is less than if the peptides are used separately for immunization.

Last update on 27.09.2011 by Matthias Reinhard.

Peptide antibodies: Can one choose a peptide that has an identical sequence in e.g. mouse and human, in order to obtain an antibody recognizing both species?

In principal this is possible, even if the sequence is identical in rabbits as well. However, this may pose an elevated risk of failure, as sequence stretches differing between various species are considered to be the better antigens. Analysis of the whole scenario (considering also additional candidate sequences) can tell whether it it worthwhile to choose a conserved peptide sequence. Most often, it is advisable to focus on a single species.

Last update on 30.10.2012 by Matthias Reinhard.

Epitope selection

Can I rely on any software for epitope prediction?

Epitope selection is a very difficult task and probaby is the most critical step of the whole project! Although much effort has been invested into the development of software for epitope prediction, the output of these programs often is even contradictory. By no means one must rely on any of these tools as the only source! Instead, our proprietary work-flow comprises a wide variety of different aspects and methods, also including 3D models (where available) and other background information.

We pre-select a set of epitope sequences, which we will discuss in detail with our customers to make the final decision.


Last update on 09.08.2011 by Matthias Reinhard.

Peptide antibodies: Is there an alternative to epitope "prediction"?

Yes, there is an alternative!

Sometimes there is an opportunity to "ask the rabbits" which epitope they have chosen in a similar previous project. Due to the availability of novel micro-scale tools it is a straightforward and very affordable method to fine map the epitope amino acid sequences of an existing serum/antibody, which is running short or which should be reproduced and fine tuned as a peptide antibody.

Please don't hesitate to ask us for more details or a quotation.

Epitope mapping with a peptide microarray
Epitope mapping with a peptide microarray: Doublet peptide spots covering the full-length protein sequence with an off-set of 1 amino acid (click to enlarge; image courtesy of Dr. V. Stadler)

Last update on 17.10.2012 by Matthias Reinhard.


How much protein do I need?

For immunization,

~1.0 mg (to 1.5 mg) of purified (recombinant or natural) protein is a very good starting point. If only small amounts of antigen are available, we can try to increase the antigenicity by various means. Thus, with as few as 30 µg of protein purified from natural sources we have already successfully run complete antibody programs.


For preparation of an affinity matrix,

3 - 5 mg of protein would be perfect. Sometimes less must be sufficient...

Last update on 09.08.2011 by Matthias Reinhard.

Do I need a certain concentration of antigen?

For immunization,

we prefer to have the antigen concentrated at about 0.5 - 1.0 mg/ml. With intradermal injections we are quite limited with volume! If your antigen is considerably more dilute, we would have to apply (at least part of it) via standard subcutan injections.


For preparation of an affinity column,

concentration is not critical. However, the solution should not be unnecessary dilute.

Last update on 09.08.2011 by Matthias Reinhard.

Are there special requirements for the buffer?

For immunization,

ideal buffers are PBS or TBS or any other physiological buffer with close to neutral pH and no toxic ingredients:

  • no sodium azide (NaN3)!
  • < 300 mM NaCl
  • < 1 mM EDTA/EGTA
  • < 1 M urea (ideally none at all)

Please keep in mind that although low amounts of detergents maybe possible, this will compromise the stability of the emulsion and result in a less efficient immunization. Therefore, please remove your detergents whenever possible!

If your protein precipitates in the absence of detergents or urea, that is no problem! We can handle that.


For preparation of an affinity matrix,

buffer requirements are less stringent, but buffers must be absolutely free of amino groups (no Tris! no glutathion!) !

Ideal buffers have a neutral to slightly alkaline pH.

Insoluble antigens (e.g. precipitates or inclusion bodies purified according to our special protocol) are no major problem. Please do not try to solubilize but leave that part to us.

Last update on 09.08.2011 by Matthias Reinhard.

Serum Collection

What serum volumes can I expect per bleed?

Here are the volumes that we guarantee and those that we actually have delivered in the past:


Type of Bleed
guaranteed actual average volume
min. - max. volume n
pre-immune ~5 ml
5.6 ml
5.0 ml - 6.0 ml
test (standard)
~3 ml
3.4 ml
3.0 ml - 4.0 ml
test (w/ affinity purification)
~5 ml
5.4 ml
5.0 ml - 6.0 ml
large (regular)
~15 ml
17.5 ml 15.0 ml - 20.0 ml

≥35-45 ml*

61.3 ml 47.0 ml - 88.0 ml

(Average and min./max. volumes of the "n" latest bleeds; numbers refer to different lengths of analysis periods backwards from 07/07/2011)
*: adjusted on 07/07/2011; previous numbers: 20-35 ml

Last update on 09.08.2011 by Matthias Reinhard.

Affinity Purification

How important is purifying the antibodies ?

1. With recombinant antigens affinity purification is often dispensable, although, you will just feel better knowing exactly what you are working with.


2. For peptide antibodies affinity purification is absolutely essential! We do not offer peptide antibodies without affinity purification, not even for testing.


a) General peptide antibodies:

If you tried already, you will know that testing crude anti-peptide sera (with myriads of antibodies against carrier proteins and adjuvants) is an unsatisfying job. Therefore, we routinely affinity purify all peptide antibodies. Judging from the yield of these purifications (mg of affinity purified antibody per ml of crude serum) we can immediately reach a conclusion about the immunization success.

b) Modification specific antibodies:

There is no way to test these antibodies in crude sera. This is due to the fact that in polyclonal sera, inevitably, there are always antibodies recognizing their corresponding site irrespective of any modification (e.g. phosphorylation or mutation). These antibodies must be removed by pre-adsorption/depletion on a second column.

In contrast, only in exceptionally rare cases there might also be (contaminating) antibodies reactive with the modification (e.g. pTyr, pSer, or pThr) irrespective of its sequence context (e.g. "pan phospho-tyrosine" antibodies). If required, these can be removed using a separate peptide/column.

Last update on 05.02.2013 by Matthias Reinhard.

I had a serum raised by some other company. Can I get it affinity purified by immunoGlobe?

Yes of course. We do not care who has raised the serum and will affinity purify it with the same diligence as if it were one of our own products.

Last update on 09.08.2011 by Matthias Reinhard.

What is a third affinity column good for?

When you want to raise an antibody against a certain modification (the modified antigen being linked to an affinity column) you need another affinity column with the unmodified antigen in order to pre-absorb/deplete those antibodies that also react with the antigen irrespective of its modification. A third column may be required to remove all antibodies that react with the modification only - independent of its context.

An example:

You raise an antibody against a certain Tyr phosphorylation site of protein X. The second column depletes all antibodies reacting also with non-phosphorylated protein X, and the third affinity coulmn will remove all antibodies reacting with phospho-Tyr in any protein.

Last update on 09.08.2011 by Matthias Reinhard.