PD016 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures.

POLYCLONAL ANTIBODY Anti-Dock6 Code No. Quantity Form PD016 100 L Affinity Purified

BACKGROUND: Small GTPases of the Rho family, SPECIES CROSS REACTIVITY: Rho, Rac, and Cdc42, are essential regulators of the Species Human Mouse Rat dynamics of actin cytoskeletal structures and diverse cellular events requiring the actin cytoskeleton. Signaling Cells 293T N1E-115 PC12 specificity of Rho GTPase pathways is achieved in part by Reactivity on WB + + + selective interaction between members of the Dbl family guanine nucleotide exchange factors (GEFs) and their Rho GTPase substrates. , also known as dedicator of INTENDED USE: cytokinesis 1 (Dock1), is an atypical GEFs for the Rho For Research Use Only. Not for use in diagnostic procedures. GTPases. It has been revealed an evolutionarily conserved superfamily with homology to Dock180 comprised REFERENCES: of at least 11 mammalian members. This family is 1) Miyamoto, Y., et al., Exp. Cell Res. 313, 791-804 (2007) structurally divided into four classes Dock-A, -B, -C, and 2) Côté, J. F., and Vuori, K., J. Cell Sci. 115, 4901-4913 (2002) -D. Dock6 belongs to the Dock-C subfamily, displays GEF activities for both Rac1 and Cdc42 and may be one of This antibody is used in reference number 1). physiological regulators of neurite outgrowth. On the other hand, member of the Dock-A and -B subfamilies except for Dock4 are Rac1-specific GEFs. Dock9 of the Dock-D 1 2 3 4 5 subfamily has specificity for Cdc42. kDa

220 - SOURCE: This antibody was purified from rabbit serum 170 - using affinity column. The rabbit was immunized with the mouse Dock6 synthetic peptide PHSLNFSSRQGSV (553-565 aa). 116 -

FORMULATION: 100 L volume of PBS containing 50% glycerol, pH 7.2. No preservative is contained. 76 -

STORAGE: This antibody solution is stable for one year from the date of purchase when stored at -20oC. 53 -

REACTIVITY: This antibody reacts with Dock6 on Western blotting. Western blot analysis of Dock6 expression in 293T (1), N1E-115 APPLICATIONS: (2), PC12 (3), mock transfectant (4) Western blotting; 1:100-1:300 for chemiluminescence and human Dock6 transfectant (5) detection system using PD016. Immunoprecipitation; Not tested Immunohistochemistry; Not tested Immunocytochemistry; Not tested* *It is reported that this polyclonal antibody can be used PROTOCOL: in Immunocytochemistry in the reference number 1). SDS-PAGE & Western Blotting Flow cytometry; Not tested 1) Subconfluent cultured cells are grown in 100-mm dishes. 2) Wash the cells 3 times with PBS. Add 400 L of Detailed procedure is provided in the following Laemmli’s sample buffer to the dish and scrape the cells PROTOCOL. into microcentrifuge tubes. 3) Boil the samples for 5 minutes and sonicate briefly (up to 10 seconds). Centrifuge the tube at 12,000 x g for 5 minutes at 4oC.

MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL https://ruo.mbl.co.jp e-mail [email protected], TEL 052-238-1904 PD016 Page 2 of 2

4) Load 20 L of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 5) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm2 for 1 hour in a semi-dry transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine, 20% MeOH). See the manufacture's manual for precise transfer procedure. 6) To reduce nonspecific binding, place the membrane in blocking buffer (10% skimmed milk, 100 mM Tri-HCl, pH 7.6, 154 mM NaCl, 0.05% Tween-20) overnight at 4oC. 7) Incubate the membrane with primary antibody diluted with blocking buffer as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 8) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times). 9) Incubate the membrane with the 1:10,000 HRP-conjugated anti-rabbit IgG (MBL; code no. 458) diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 10) Wash the membrane with PBS-T (10 minutes x 3 times). 11) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute. 12) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 13) Expose to an X-ray film in a dark room for 3 minutes. 14) Develop the film as usual. The condition for exposure and development may vary.

(Positive controls for Western blotting; 293T, N1E-115, PC12)

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