OriGene Technologies, Inc. OriGene Technologies GmbH

9620 Medical Center Drive, Ste 200 Schillerstr. 5 Rockville, MD 20850 32052 Herford UNITED STATES GERMANY Phone: +1-888-267-4436 Phone: +49-5221-34606-0 Fax: +1-301-340-8606 Fax: +49-5221-34606-11 [email protected] [email protected]

AM26545AF-N Monoclonal Antibody to SIPA1 / SPA1 - Azide Free

Alternate names: GTPase-activating Spa-1, Signal-induced proliferation-associated protein 1, Sipa-1, Spa-1, p130 SPA-1 Quantity: 0.1 mg Concentration: 1.0 mg/ml Background: SPA-1 , signal-induced proliferation associated 1, encodes a 130 kDa protein (p130 SPA-1 ) with a domain related to GTPase activating protein (Rap1GAP) at the N-terminus and to PDZ protein interaction domain followed by a leucine zipper motif at the C-terminus. It exhibits a specific GAP activity for Ras-related regulatory Rap1 and Rap2, but not for other small GTPases. p130 SPA-1 was mostly localized at both the plasma membrane and the perinuclear membranous region co- localizing with Rap1 and Rap2. Expression of SPA-1 and rap1GAP tended to be segregate in various tissues, lymphoid and hematopoietic tissues expressing abundant SPA-1 transcript without rap1GAP , while those such as brain, kidney and pancreas exhibiting rap1GAP mRNA with little SPA-1 . Uniprot ID: P46062 NCBI: 10090 Host / Isotype: Mouse / IgG2a Clone: 2-22-9 Immunogen: Wister rat splenocyte immunized with the baculoviral expressed recombinant mouse SPA-1 Format: State: Liquid Ig fraction Purification: Anion exchange column chromatography Buffer System: PBS containing 50% glycerol, pH 7.2. No preservative is contained. Applications: Western blot: 1µg/ml for chemiluminescence detection system. Immunoprecipiation: 5 µg/300 ml of cell extract from 1x 107 cells. For details see protocols below. Other applications not tested. Optimal dilutions are dependent on conditions and should be determined by the user. Specificity: This antibody reacts with SPA-1. Species Reactivity: Tested: Mouse (Ba/F3, NIH/3T3, WR19L, MEF), Hamster (CHO). Does not react with Human (HL-60, Jurkat, Raji) or Rat (Rat-1). Add. Information: This product was originally produced by MBL International. Storage: Upon receipt, store (in aliqouts) at -20 °C. Avoid repeated freezing and thawing. Shelf life: One year from despatch. General Readings: 1. Kometani, K., et al., Cancer Res. 66 , 9967-9975 (2006). 2. Jin, A., et al., Oncogene 25 , 4332-4340 (2006). 3. Farina, A., et al., Mol. Cell. Biol. 24 , 9059-9069 (2004). 4. Ishida, D., et al., PNAS . 100 , 10919-10924 (2003).

For research and in vitro use only. Not for diagnostic or therapeutic work. Material Safety Datasheets are available at www.acris-antibodies.com or on request. OG/20130815 1 / 3 AM26545AF-N: Monoclonal Antibody to SIPA1 / SPA1 - Azide Free

5. Su, L., et al., J. Biol. Chem. 278 , 15232-15238 (2003). 6. Tsukamoto, N., et al., J. Biol. Chem. 274 , 18463-18469 (1999). 7. Kurachi, H., et al., J. Biol. Chem. 272 , 28081-28088 (1997). 8. Hattori, M., et al., Mol. Cell. Biol. 15 , 552-560 (1995). Protocols: SDS-PAGE & Western Blotting 1) Wash the 1 x 10e7 cells 3 times with PBS and suspend with 1 mL of Laemmli’s sample buffer, then sonicate briefly (up to 20 seconds). 2) Boil the samples for 3 minutes and centrifuge. Load 20 µL of the sample per lane in a 1 mm thick SDS-polyacrylamide gel for electrophoresis. 3) Blot the protein to a polyvinylidene difluoride (PVDF) membrane at 1 mA/cm 2 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. 4) To reduce nonspecific binding, soak the membrane in 10% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature, or overnight at 4oC. 5) Incubate the membrane with primary antibody diluted with PBS, pH 7.2 containing 1% skimmed milk as suggest in the APPLICATIONS for 1 hour at room temperature. (The concentration of antibody will depend on condition.) 6) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 minutes x 3 times). 7) Incubate the membrane with the 1:10,000 HRP-conjugated anti-rat IgG diluted with 1% skimmed milk (in PBS, pH 7.2) for 1 hour at room temperature. 8) Wash the membrane with PBS-T (10 minutes x 3 times). 9) Wipe excess buffer on the membrane, then incubate it with appropriate chemiluminescence reagent for 1 minute. 10) Remove extra reagent from the membrane by dabbing with paper towel, and seal it in plastic wrap. 11) Expose to an X-ray film in a dark room for 3 minutes. 12) Develop the film as usual. The condition for exposure and development may vary. (Positive controls for Western blotting; NIH/3T3, WR19L, MEF, CHO)

Immunoprecipitation 1) Wash the cells 3 times with PBS and suspend with 10 volume of cold Lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% NP-40) containing appropriate protease inhibitors. Incubate it at 4oC with rotating for 30 minutes, then sonicate briefly (up to 10 seconds). 2) Centrifuge the tube at 12,000 x g for 10 minutes at 4oC and transfer the supernatant to another tube. 3) Add primary antibody as suggest in the APPLICATIONS into 300 µL of the supernatant. Mix well and incubate with gentle agitation for 30-120 minutes at 4oC. 4) Add 20 µ L of 50% protein G agarose beads resuspended in the cold Lysis buffer. Mix well and incubate with gentle agitation for 60 minutes at 4oC. 5) Wash the beads 3-5 times with the cold Lysis buffer (centrifuge the tube at 2,500 x g for 10 seconds). 6) Resuspend the beads in 20 µ L of Laemmli’s sample buffer, boil for 3-5 minutes, and centrifuge for 5 minutes. Use 10 µL/lane for the SDS-PAGE analysis. (See SDS- PAGE & Western blotting.) (Positive control for Immunoprecipitation; NIH/3T3)

For research and in vitro use only. Not for diagnostic or therapeutic work. Material Safety Datasheets are available at www.acris-antibodies.com or on request. OG/20130815 2 / 3 AM26545AF-N: Monoclonal Antibody to SIPA1 / SPA1 - Azide Free

Pictures: Western blot analysis of mouse SPA-1 expression in NIH/3T3 (1), WR19L (2), CHO (3) and MEF (4) using AM26545AF-N.

Immunoprecipitation of mouse SPA-1 from NIH/3T3 with rat IgG2a (1) or AM26545AF-N (2). After immunoprecipitated with the antibody, immunocomplex was resolved on SDS- PAGE and immunoblotted with AM26545AF-N.

For research and in vitro use only. Not for diagnostic or therapeutic work. Material Safety Datasheets are available at www.acris-antibodies.com or on request. OG/20130815 3 / 3

Powered by TCPDF (www.tcpdf.org)