FREQUENTLY ASKED QUESTIONS

Core Home | Summary of services | Price list | Antigen information | Work order

How long does it take to make an antibody?

Rabbit Polyclonal Antibody usually takes 4 - 5 months.  We usually perform 5 – 6 injections at 3 week intervals with bleeds 10 – 14 days post injection.

Mouse Polyclonal Antibody usually takes about 3 months.  We usually perform 5 injections at 2 week intervals with bleeds 10 – 12 days post injection.

Mouse Monoclonal Antibody takes the 3 months needed to make the polyclonal antibody plus an additional month or two for screening and single cell cloning.

 

How much serum/antibody will be produced?

Rabbit Polyclonal Antibody bleeds typically yield 10 – 15 ml serum/rabbit on a routine bleed and 50 – 80 ml/rabbit upon exsanguination. Since we use 2 rabbits per antigen, this is a considerable amount of polyclonal serum

Mouse Polyclonal Antibody bleeds typically yield 100 – 150 µl serum/mouse on a routine bleed and 300 – 500 µl/mouse upon exsanguination.  Although we use 3 mice per antigen, the cumulative amount is quite small.

Mouse Monoclonal Antibody is secreted by hybridoma cells thus the yield can be infinite.

 

How does the antigen need to be prepared?

We usually inject fusion proteins such as GST or His so protein can be given to us in eluted form or bound to beads. We can also inject peptides conjugated to KLH or BSA.

The cleaner the protein, the better the antibody!  If protein is eluted, it must be dialyzed into a biological buffer without detergent or Urea.  If protein is still bound to beads, beads must be washed thoroughly with a biological buffer.  Eluted protein will be mixed with Freund’s Adjuvant before injection.  Freund’s Adjuvant can cause distress and discomfort to the animal so we prefer to inject antigen bound to beads.

 

We also prefer the antigen to be as close to 2 mg/ml as possible because the maxium injection volumes are 500 µl (rabbits) and 150 µl (mice).  Remember, eluted antigen gets mixed with equal volume Freund’s Adjuvant and bound antigen is usually injected as a 50% slurry thus making the 2 mg/ml concentration 1 mg/ml for injection.

 

How much antigen is needed for antibody production?

Rabbit Polyclonal Antibody
2 rabbits/antibody x 400 - 500 µg antigen/rabbit = 800 µg - 1 mg antigen/boost
5 boosts x 800 µg - 1 mg antigen/boost = 4 – 5 mg antigen

Mouse Polyclonal/Monoclonal Antibody

3 mice/antibody x 100 µg antigen/mouse = 300 µg antigen/boost
4 -5 boosts x 300 µg antigen/boost = 1.2 – 1.5 mg antigen

If making monoclonal antibody, an additional 1 mg of antigen is needed for ELISA screening.

 

NOTE:  Although the above antigen amounts are preferred, we can get away with less antigen if and when it is difficult to produce.  We can also start the injections without the total antigen amount, but it is imperative that we have enough for at least 2 – 3 injections as well as a guarantee that the remaining antigen will be given to us in a timely fashion.

 

What are the most common problems with antigen preparation?

Some fusion proteins are not expressed well for various reasons.  It is best to try different induction times, temperatures, and concentrations in order to find the optimal expression of your protein.  If this doesn’t work, you can try subcloning into another vector to see if you can increase your yield by using another method.

It is also quite common that fusion proteins are insoluble.  Sometimes, varying induction time, temperature, or concentration can decrease the amount of insoluble protein, but expression may also decrease.  There are many protocols available for purifying insoluble protein.  It doesn’t really matter which protocol is used, just remember that most protocols for solubilizing insoluble protein contain denaturing substances such as Urea or detergent.  Although these protocols usually have renaturing steps, the protein doesn’t always refold correctly often times making it difficult to get antibody to the native form of the protein.  This doesn’t mean you won’t get a good antibody, it just means that you may not get the antibody you need.  For common applications such as Western blotting, an antibody need only recognize the denatured form of the antigen.  For other applications (eg. antibody supershift, immunoprecipitation), the antibody must recognize the native form of the antigen.  However, even polyclonal antibodies made against entirely denatured antigen usually recognize some epitopes on the native form.

 

Rabbit Polyclonal v. Mouse Polyclonal v.  Mouse Monoclonal antibodies?

In general, if you need a lot of antibody and quickly, rabbit polyclonal antibody is the way to go.  Just remember it is in finite supply and it can be rather dirty.  The best way to clean up your antibody is to affinity purify it, but this will require more antigen.  Other methods can also be used to clean up the rabbit serum. In slightly less time than it takes to make rabbit polyclonal antibody, you can make mouse polyclonal antibody.  Although it is typically very clean, there is very little supply so this is usually used in conjuction with making hybridomas for mouse monoclonal antibody.  If you have the time and money to invest, mouse monoclonal hybridoma cell lines will provide you with an endless supply of antibody that is usually quite clean.  Mouse monoclonal antibody that has been single cell cloned also gives you the ability to isolate antibodies to specific epitopes. All of these are viable options depending on your intended use.  Contact us for more information if you are having trouble deciding which antibody will be best for your needs.