imagej western blot quantification protocol

Mayer BJ, Jackson PK, Van Etten RA, Baltimore D (1992) Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.Smith DB, Johnson KS (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.Cold Spring Harbor Press, Cold Spring Harbor, NY, pp 37–57 In: Golemis E (ed) Protein-protein interactions. Einarson MB, Orlinick JR (2002) Identification of protein-protein interactions with glutathione-S-transferase fusion proteins.Burnham MR, DeBerry R, Bouton AH (2001) Detection of phosphorylation-dependent interactions by far-western gel overlay.Moorhead G, MacKintosh C (2004) Affinity methods for phosphorylation-dependent interactions.Machida K, Thompson CM, Dierck K, Jablonowski K, Karkkainen S, Liu B, Zhang H, Nash PD, Newman DK, Nollau P, Pawson T, Renkema GH, Saksela K, Schiller MR, Shin DG, Mayer BJ (2007) High-throughput phosphotyrosine profiling using SH2 domains.Nollau P, Mayer BJ (2001) Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.Blackwood EM, Eisenman RN (1991) Max: a helix-loop-helix zipper protein that forms a sequence-specific DNA-binding complex with Myc.Macgregor PF, Abate C, Curran T (1990) Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BP1.Cicchetti P, Mayer BJ, Thiel G, Baltimore D (1992) Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho.Hall RA (2004) Studying protein-protein interactions via blot overlay or Far Western blot.Akiyama T, Ohuchi T, Sumida S, Xu SQ, Toyoshima K (1992) Phosphorylation of the anti-oncogene products and control of the cell cycle.Hoeffler JP, Lustbader JW, Chen CY (1991) Identification of multiple nuclear factors that interact with cyclic adenosine 3′,5′-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions.In: Coligen JE (ed) Current protocols in protein science, vol 2. Edmondson DG, Dent SY (2001) Identification of protein interactions by far Western analysis.We also present a batch quantification method that allows for the direct comparison of probe binding patterns.

We here describe a rapid and simple protocol for far-western blotting, in which GST-tagged Src homology 2 (SH2) domains are used to probe cellular proteins in a phosphorylation-dependent manner. When specific modular protein binding domains are used as probes, this approach allows characterization of protein–protein interactions involved in biological processes such as signal transduction, including interactions regulated by posttranslational modification. In contrast to western blotting, which uses specific antibodies to detect target proteins, far-western blotting detects proteins on the basis of the presence or absence of binding sites for the protein probe. Far-western blotting is a convenient method to characterize protein–protein interactions, in which protein samples of interest are immobilized on a membrane and then probed with a non-antibody protein.

Imagej western blot quantification protocol