Introduction | Table 1 | Figure 1 | Figure 2

Isoform specific antibodies were raised against human ARF1, ARF3, ARF4, ARF5, and ARF6 and described in Cavenagh, et al (1996). This page summaries the characterization of the peptide antisera described in that paper and used in our work (e.g., see, and that of others. These reagents have been given to more than 100 researchers during the past 10 years. As a result, our supplies are now in more limited supply and can only be shared after discussion with Dr. Kahn.

Table I summarizes the names of the antisera for each Arf isoform, the recommended dilutions for use in immunoblotting, and the observed cross-reactivity amongst isoforms. Each of the polyclonal antisera were raised against peptides, conjugated to keyhole limpet hemocyanin (KLH), derived from the sequences of the human Arf proteins, as described in Cavenagh, et al (J. Biol. Chem. ,271: 21767-21774, 1996).

Recognition of the whole protein and specificity of each antiserum for one isoform are indicated by the slot blot, shown in figure 1. Note that the anti-Arf4 antibody, R-891, recognizes Arf5 in this slot blot. Only the Arf4 staining is specific as it is blocked by the immunizing peptide. Standard dilutions, sensitivity in immunoblots, and cross-reactivities are indicated in Table I. Note that the polyclonal antibodies are used as unfractionated serum. As such, bands migrating with different apparent molecular weights (away from the low 20 kDa range) are often observed when blotting whole cell or tissue lysates. Experience indicates that these bands are not blocked by the immunizing peptide and are thus considered non-specific.

Figure 2 illustrates the use of these antisera against whole tissue lysates. Levels of Arf1, 3 and usually 4 are sufficient to detect with these reagents. Note also that the anti-Arf4 antiserum, R-891, is more sensitive than the others so even though a signal is clearly seen in the immunoblots shown in fig. 2, the actual levels of Arf4 are less than those of Arf3. Results of Northern blotting indicate that the message of each Arf is present in every sample tested. This suggests that the protein levels are often below the lower limit of detection and cannot be interpreted as lack of expression. Protein sequences of Arf1, Arf3, Arf5, and Arf6 are identical or very nearly so amongst mammalian species. Differences in the sequences of human Arf4 and other mammalian species result in poor reactivity of the Arf4 antiserum, R-891, with rodent, bovine, and likely Arf4 from other species. Interestingly, this antiserum (R-891) is the only of the peptide antisera that is useful for indirect immunofluorescence, even for rodent cells in culture. This immunoreactivity is specific as it is blocked by the immunizing peptide and the (perinuclear) localization is sensitive to brefeldin A.

The monoclonal antibody 1D9 is described on its own page. It is included in the immunoblotting here for comparative purposes. Unless specific information regarding isoforms is required, 1D9 is the reagent of choice for most uses.

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Fig. 1. Specificity of the Arf antibodies is illustrated in slot blots of purified recombinant Arf proteins with isoform-specific and monoclonal anti-Arf antibodies. The five human Arf proteins used are shown on the top in a Coomassie Blue-stained SDS-polyacrylamide gel loaded with 0.3 µg/lane. Each antibody was tested for immunoreactivity against each of the different Arf proteins in slot blots. Antisera were diluted as indicated in Table I. Note that the cross-reactivity of the Arf4 antiserum (R-891) with Arf5 is nonspecific as it is not competed with the immunizing peptide.

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Fig. 2. Proteins from eight human tissues were tested for immunoreactivity with the isoform-specific and monoclonal anti-Arf antibodies. Tissue lysates (25 µg/lane) were analyzed by immunoblotting.Positive controls (15 ng) for each Arf isoform are included on the left. Note the differences in sensitivity among the different antibodies, indicated in Table I.

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