We all know that antibodies can be made using non-conventional means by injecting proteins that are adsorbed (non-covalent or covalently linked) to various solid surfaces (Nitrocellulose, Sepharose) or embedded in SDS-PAGE gels. Nitrocellulose has been used occasionally but it is not recommended unless there are no other options. Proteins bound to Sepharose (GST/MBP/His fusion proteins) and SDS-PAGE have proven more useful in making antibodies . There are certain guidelines that should be kept in mind to assure the survival of animal and generation of antibodies. The advantages and disadvantages of these methods (please see below) should be considered as well.

One must remember that quality of antibodies depends upon the quality of antigens. What Goes In Determines What Comes Out (WGIDWCO). Animals will make antibodies to anything that is foreign to them (GST/His tag/MBP, proteins of interest or whatever). It is immaterial to an animal whether it is a protein of interest or not or your whole grant proposal or the outcome of research paper approval depends upon making antibodies to "your protein". Animals couldn’t care less if you make antibodies or not. If fact, if given a choice, they would rather not be subjected to the process of making antibodies. Most bacterial or viral proteins are much more immunogenic than mammalian proteins. Very often, a strong immune response may be evoked by even minute concentration of bacterial protein contaminants present in the antigen. In most cases this does not create a problem since the bacterial antigens are not present in most mammalian protein cells. Even if anti-bacterial proteins becomes problematic, it is possible to remove them by either solid phase affinity adsorption or simply by adding these antigens to antibody solution.

Proteins Bound to various affinity matrices or SDS-PAGE gels.

The idea is to inject as much antigen as possible with a minimum amount of gels or beads. After all, animal do need to get rid of the gels and beads. If you inject too much gels or beads, the animal may die during the project. Acrylamide is neurotoxic even at minimal doses. A small percentage of gels remains unpolymerized even after electrophoresis (Gloves must be worn at all times when making or handling gels). In a comparative study, we found that the gels that are unstained are less tolerated by animals than the ones that have been fixed, washed, and stained (the process may remove lots of undesirable elements from the gels including Acrylamide, SDS, etc.). Unless you have reasons not to stain the gel, stain the gel and cut out the band of interest. Too much Sepharose (>0.25 ml packed beads per injection per rabbit) has been problematic and creates persistent wounds at the injection site. Please keep these points in minds:

1. Try processing required protein sample on just 1 gel. This amount should be enough for the entire project in 1 or 2 rabbits (5-8 injections over 2 months). It is a good idea to inject 25-100 ug/per injection/per rabbit. One gel should have enough protein loaded for up to 10-15 injection for both rabbits. Load up to 2 mg total protein per gel. Protein resolution is not of prime importance.

2. Run Minigels or std. Gels (1 mm thickness). Use a prep-comb (1 big flat comb with no wells). Stain the gels with coomassie blue, destain, and cut the band of interest using the whole width of the gels.

3. Gels can be sent at room temperature in a tube. Homogenize the gels to a point that it can pass through an 20-22 g needles for injections. Each injection should contain an approx. amounts of 25-100 ug.

4. Try spreading the gels at several sites (4-6 subcutaneous and 2 Intramuscular) to decrease the chances of inducing non-desirable reaction. Do not inject less than 2 weeks apart.

5. Do not inject more than 100-200 ul packed beads at spread them over to several sites. Beads should be washed with PBS to remove all buffers or detergents.

6. It is a good idea to mix the beads with incomplete Freund’s adjuvant. It helps to make a uniform emulsion for injection and further increase antigenicity.

7. If soluble protein becomes available during immunization, it should be included in subsequent injections to increase the quality and quantity of antibody.

Advantages and Disadvantages of using gels/beads for antibody production

These methods should only be used when purified material is not available or difficult to obtain. Antibodies generated by this methods may be low titer because of limited antigen dosages. Antibodies may only react with the denatured protein and may not immunoprecipitate native proteins. However, this methods does offer the unique advantage of making antibodies without extensive purification of antigens. Antibodies can be generated to closely related proteins that are difficult to separate/purify. This method should not be used because some recombinant antigens after elution from some affinity columns (e.g., Nickle-Sepharose) remain insoluble in most common buffers unless 4M or higher Urea is present. For most antibody generation purpose, we can use proteins in urea if protein concn is kept sufficiently high (2-10 mg/ml; higher the better to reduce volume of urea containing buffers). It is also possible to inject proteins as precipitates or suspension. Therefore, protein insolubility should not be the overriding factor in using SDS-gels. Protein in solutions, even if in Urea or in precipitated forms, are better than using SDS-gels.