PD043 Page 1 For Research Use Only. Not for use in diagnostic procedures. Anti-Phospho-SF3B1 (Sap155) (Ser129) pAb CODE No. PD043 CLONALITY Polyclonal ISOTYPE Rabbit Ig, affinity purified QUANTITY 100 L SOURCE Purified IgG from rabbit serum IMMUNOGEN KLH conjugated synthetic peptide, RTMII(pS)PERL (corresponding to amino acid residues 124-133 of human SF3B1) FORMURATION PBS containing 50% Glycerol (pH 7.2). No preservative is contained. STORAGE This antibody solution is stable for one year from the date of purchase when stored at -20°C. APPLICATION-CONFIRMED Western blotting 1:1000 for chemiluminescence detection system SPECIES CROSS REACTIVITY on WB Species Human Mouse Rat Hamster Cell HeLa, Raji NIH/3T3, WR19L Not tested Not tested Reactivity Entrez Gene ID 23451 (Human), 81898 (Mouse) REFERENCES 1) Wang, C., et al., Genes Dev. 12, 1409-1414 (1998) 2) Shi, Y., et al., Mol Cell 23, 819-829 (2006) 3) Tanuma, N., et al., J. Biol. Chem., 283, 35805-35814 (2008) 4) Yoshida, K., et al., Nature 478, 64-69 (2011) 5) Rossi, D., et al., Blood 118, 6904-6908 (2011) 6) Quesada, V., et al., Nat Genet 44, 47-52 (2011) 7) Wang, L., et al., N Engl J Med. 365, 2497-2506 (2011) 8) Biankin, A. V., et al., Nature 491, 399-405 (2012) For more information, please visit our web site http://ruo.mbl.co.jp/ MEDICAL & BIOLOGICAL LABORATORIES CO., LTD. URL http://ruo.mbl.co.jp/ e-mail [email protected], TEL 052-238-1904 PD043 Page 2 P a g RELATED PRODUCTS e PD043 Anti-Phospho-SF3B1 (Sap155) (Ser129) pAb D138-3 Anti-Sap155 (SF3B12) mAb D221-3 Anti-Sap155 (SF3B1) mAb D139-3 Anti-Ring1B (Mouse) mAb RN068PW Anti-PPP1R8 pAb RN085PW Anti-U2AF1 pAb RN086PW Anti-U2AF2 pAb D309-3 Anti-IDH1 mAb (RMab-3) D331-3 Anti-IDH1-R132G (Human) mAb (GMab-r1) D299-3 Anti-IDH1-R132H (Human) mAb (HMab-1) D300-3 Anti-IDH1-R132S (Human) mAb (SMab-1) D311-3 Anti-IDH2 mAb (RMab-22) D330-3 Anti-IDH2 mAb (KrMab-3) D328-3 Anti-IDH2-R172K (Human) mAb (KMab-1) PD043 Page 3 SDS-PAGE & Western blotting 1) Wash 1 x 107 cells 3 times with PBS and suspend with 1 mL of Laemmli’s sample buffer, then sonicate briefly (up to 10 sec.) 2) Centrifuge the tube at 12,000 x g for 5 min. at 4°C and transfer the supernatant to another tube. 3) Boil the samples for 3 min. and centrifuge at 12,000 xg for 5 min. Load 10 L of the sample per lane in a 4-15% Mini-PROTEAN® TGXTM Precast Gel (Bio-Rad; code no. 456-1085) and carry out electrophoresis. 4) Blot the protein to a nitrocellulose membrane at 60 V for 4 hr. at 4°C in a wet transfer system (Transfer Buffer: 25 mM Tris, 190 mM glycine). See the manufacturer's manual for precise transfer procedure. 5) To reduce nonspecific binding, soak the membrane in 5% skimmed milk (in PBS, pH 7.2) for 20 min. at room temperature. 6) Incubate the membrane with primary antibody diluted with Can Get Signal-1 (TOYOBO; code no. NKB-201) as suggested in the APPLICATION for 1 hr. at room temperature. (The concentration of antibody will depend on the conditions.) 7) Wash the membrane with PBS-T [0.05% Tween-20 in PBS] (5 min. x 3 times). 8) Incubate the membrane with the 1:3000-5000 of goat anti-rabbit IgG-HRP (Santa Cruz; code no. sc-2004) diluted with Can Get Signal-2 (TOYOBO; code no. NKB-201) for 20 min. at room temperature. 9) Wash the membrane with PBS-T (10 min. x 3 times) 10) Wipe excess buffer on the membrane, and then incubate it with appropriate chemiluminescence reagent for 1 min. 11) Expose for 3 min. with ImageQuant LAS 4000 imaging system (Fujifilm). The condition for exposure may vary. (Positive controls for Western blotting; HeLa) 1 2 Western blot analysis of phospho-SF3B1 (Ser129) from HeLa cells 1: Anti-Phospho-SF3B1 mAb (Ser129) (PD043) 2: Anti-SF3B1 mAb (MBL; code no. D221-3) This data and protocol were provided by Dr. Tanuma, Miyagi Cancer Center Research Institute 130326-1 .