Use of Mouse Monoclonal 1D9, anti-ARF Antibody and Isoform Specific pAbs
The full name of this antibody is 1D9-1E10-1C6, but is always abbreviated to 1D9 without confusion. This is a mouse IgG1 class antibody raised against purified human ARF1 protein. The antibody can be obtained from Affinity Bioreagents, catalog MA3-060 or other retailers. Alternatively, you can enquire into collaborative projects with the Kahn lab. Characterization of this antibody was described in Cavenagh, et al (1996) “Intracellular distribution of Arf proteins in mammalian cells: Arf6 is uniquely localized to the plasma membrane”, J. Biol. Chem., 271:1767-21774.
Specificity:
The specific epitope recognized by this monoclonal is still not known, though it appears to include lysine 72. It recognizes human ARF1, ARF3, ARF5, and ARF6 well but human ARF4 is ~20 fold less reactive. It does not detect the ARL proteins, e.g. ARL1, ARL2, or ARL3 by immunoblotting. As ARF1 is well conserved in evolution this antibody is also good for detection of at least one species of ARF in mouse, rat, other mammals, and some yeast but not S. cerevisiae.
Use in Immunoblotting:
The 1D9 antibody is generally used at a final concentration of 1-10 μg/ml as primary antibody in an immunoblot and ECL detection. The lower limit of detection in our lab is 0.1-10 ng ARF protein per lane. Note: IgG1 does not bind well to protein A.
Use in immunofluorescence:
The antibody works well after a dilution of 1/100-1/500 in our standard cell staining protocol. Cells are generally grown on coverslips to ~70% confluence, fixed in 2% formaldehyde in PBS, and permeabilized with 0.2% saponin prior to antibody addition.
Use in immunoprecipitation:
1D9 does work to IP Arfs from solution but from mammalian cells or tissues appears to require detergents (e.g. RIP-A buffer). Thus, protein-protein interactions and bound nucleotides do not survive well, though the former should after cross-linking.
Storage:
The antibody is quite stable at 4°C, with sodium azide, although if it will not be used for long periods of time freezing is recommended. Avoid repeated cycles of freezing and thawing.
Use of Isoform-Specific Arf Antibodies
Isoform Rabbit ID Dilution (for Westerns) Notes
Arf1 R-1026 1:500
Arf3 R-1023 1:2000 detects ~20ng recombinant Arf1, but much weaker than Arf3
Arf4 R-891 1:1000 almost equally recognizes Arf4 and Arf5, but Arf5 runs higher in gels
Arf5 R-1525 1:250 detects ~20ng Arf4, but much weaker than Arf5, and Arf5 runs higher
Arf6 R-1471 1:1000 recognizes both Arf6 and Arf5, but Arf5 runs higher.
could detect 20ng arf4, arf4 runs between arf5 and arf6
*The stocks in –20 freezer have been diluted 1:1 in glycerol