Mouse anti his tag monoclonal antibody

Protein isolation was performed as previously described [41 (link)]. Briefly, cells were lysed with lysis buffer containing 50 mM Tris-HCl, 1% SDS, 1 mM dithiothreitol (DTT), and 0.43 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (ABSF). The lysates were sonicated and centrifuged at 20,400× g at 4 °C for 20 min, and the supernatants were collected. Equal amounts of protein extract were subjected to SDS-PAGE and transferred to a PVDF (polyvinylidene difluoride) membrane (EMD Millipore, Billerica, MA, USA). The following were used as the primary antibodies: Mouse anti-human thymidylate synthase monoclonal antibody (ab58287; Abcam, Cambridge, UK), mouse anti-human β-actin monoclonal antibody (A5441; Sigma), mouse anti-human α-tubulin monoclonal antibody (CP06; CALBIOCHEM), mouse anti-human ANT2 polyclonal antibody (H00000292-B01P; Novus Biologicals, LLC, Centennial, CO, USA), rabbit anti-human PHB2 monoclonal antibody (#14085; Cell Signaling Technology, Danvers, MA, USA), mouse anti-DDK(FLAG) monoclonal antibody (TA50011-100; Origene), and mouse anti-His-Tag monoclonal antibody (#2366; Cell Signaling Technology). Signals were detected with Chemi-Lumi One L (Nacalai Tesque, Kyoto, Japan) or ImmobilonTM Western Chemiluminescent HRP Substrate (EMD Millipore).

Cells were lysed with buffer containing 50 mM Tris-HCl, 1% SDS, 1 mM dithiothreitol (DTT), and 0.43 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (ABSF). The lysates were sonicated and centrifuged at 20,400 g at 4°C for 20 min, and the supernatants were collected. Equal amounts of protein extract were subjected to SDS-PAGE and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). Primary antibodies were as follows: rabbit anti-p44/42 MAPK (ERK1/2) (#9102), rabbit antiphospho-p44/42 MAPK (ERK1/2) (#9101), rabbit anti-PARP (#9542), rabbit anti-Puma (#4976), rabbit anti-p53 (#9282), mouse anti-His-Tag monoclonal antibody (#2366; Cell Signaling Technology, Beverly, MA, USA), rabbit anti-BIM (ab32158), rabbit anti-RPS5 (ab58345, Abcam), mouse anti–β-actin monoclonal antibody (A5441, Sigma), mouse anti-GAPDH (#5G4, HyTest Ltd., Turku, Finland), and mouse antihuman α-tubulin monoclonal antibody (CP06, Calbiochem, La Jolla, CA, USA). Signals were detected with Chemi-Lumi One L (Nacalai Tesque) or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore).