1. This high-ionic-strength buffer, described in ref. 9, is important for efficient extraction and solubilization of the IRS proteins. In our laboratory, Triton X-100 yields improved solubilization in comparison to NP-40-based lysis buffers. In addition, the high-salt content of the buffer is essential to successful extraction of IRS proteins from 3T3-L1 adipocytes. In this cell type, IRS is reported to maintain tight association with the cytoskeleton, and the high-salt concentration likely aids in the dissociation of IRS proteins from cytoskeletal elements (10).

2. Because 3T3-L1 adipocytes do not express IRS-3 nor IRS-4, only methods to assay IRS-1 and IRS-2 tyrosine phosphorylation are detailed here (11,12). Antibodies that selectively recognize IRS-3 and IRS-4 are available from Upstate Biotechnology.

3. Titrate the antibodies empirically for each cell line/tissue, quantity of protein, and antibody of choice to determine ideal immunoprecipitation conditions. These assays also work if the immunoprecipitating antibody recognizes phosphotyrosine; however, this precludes the opportunity to probe for equivalent immunoprecipita-tion of total IR or IRS protein among all the samples. Immunoprecipitations can be performed overnight at 4°C; however, some loss of phosphorylation may result.

4. If lysate is limiting or the protein of interest is expressed at very low levels, the entire immunoprecipitated sample can be loaded on one gel. The blot should be probed first for phosphotyrosine signal, as the phosphates are often labile. The blot can be stripped effectively using a reagent such as Restore™ from Pierce. It can then be reblocked and reprobed with antibodies to detect total IR or IRS.

5. The simplest way to detect IR or IRS tyrosine phosphorylation is to run 20-30 ig total cell lysate samples on a protein gel and probe the blot using phosphotyrosine antibodies.

6. The blots can be incubated in blocking solution overnight at 4°C.

7. The HRP-PY99 antibody, which does not require a secondary antibody, yields very clean phosphotyrosine blots. However, it is also a less sensitive mode of detection because it does not employ the typical amplification step that is intrinsic to the addition of HRP-conjugated secondary antibodies. If the phosphotyrosine signal is weak, it is better to use a simple anti-phosphotyrosine primary antibody (e.g., PY20, 4G10, PY99) and then perform the standard secondary antibody incubation with an HRP-anti-mouse-IgG antibody. In addition, choosing the most sensitive type of film available will also help in successful signal detection (e.g., X-OMAT AR from Kodak). The blots can be incubated in primary antibody overnight at 4°C; however, the blots should be developed as quickly as possible to prevent loss of the phosphorylated tyrosines.

8. Incubating the blots in secondary HRP-conjugated antibodies for longer than 2 h increases background intensity.

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