It is rare for recombinant proteins to be the same molecular weight as their natural counterpart. Sequences are either deleted or added as a function of the restriction enzyme sites in the nucleic acid or the expression vector used to produce the proteins. Additionally, there are sometimes oligomers and partial cleavage products. Neither affects the function, but sometimes they will cause confusion. To alleviate doubt, use a monoclonal antibody to demonstrate the protein in a Western blot or ELISA. Please see the specification sheet for each protein for its correct molecular weight.

All ABI proteins work in Western blot according to the considerations mentioned for ELISA. For reference use, ABI uses the following procedure for Western blot (to be developed with monoclonal antibodies):

The antigen is electrophoresed on a 4 - 15% polyacrylamide minigel at a density of approximately 4 µg of protein per centimeter of gel width and transferred to PVDF. The membrane is blocked with 1% calfskin gelatin/5% heat inactivated Normal Goat Serum in PBS/0.05% Tween 20 and allowed to air dry while suspended. The membrane is then cut into 3 mm strips, and one strip is incubated with one milliliter of 10 µg/ml (1:100) antibody in blocking buffer for two hours at 37°C. The strip is washed in three changes of PBST20 for five minutes each and incubated with 1 milliliter of diluted Goat anti-mouse IgG-AP (BioRad 170-6520) in 100 mM Tris 150 mM NaCl pH 8.0 supplemented with 5% heat inactivated Normal Goat Serum for one hour at 37°C. The strip is washed as before and developed with BCIP/NBT (BioRad 170-6432) per manufacturer's instructions.

All ABI proteins work in ELISA; however, it is critical to use an appropriate primary antibody and conjugate dilution. Double-block titrations should be performed to determine the optimum use dilutions in your system.

Natural proteins are made in infected cells using cell culture. Recombinant proteins are made in various systems, generally yeast or baculovirus. All are screened for function in ELISA and Western blot prior to selection.

We recommend that all proteins be aliquoted into use-size portions and stored frozen at -70°C or below. Avoid multiple freeze-thaw cycles as this will affect protein activity. Do not dilute and freeze--make final dilution just prior to use.

All of the proteins, whether natural or recombinant, are purified to greater than 95% as judged by Coomassie Blue stained SDS-PAGE. Purification methodology varies according to the individual product requirements, as do the buffers in which the proteins are frozen.

All ABI proteins are supplied frozen on dry ice.