b'AntibodiesWe are continually updating our Invitrogen primary antibody portfolioto either the N-terminus or C-terminus of a protein. If no antibody binds with advanced and verified products for use in flow cytometryto the protein of interest, adding a Myc tag to the protein and selecting (FC), immunohistochemistry (IHC), immunofluorescence (IF),an antibody that targets the Myc epitope can be a reliable way to immunocytochemistry (ICC), western blotting (WB), enzyme-linkeddetect the protein. Common applications include immunoprecipitation immunosorbent assays (ELISAs), and other applications. (IP), IF, and WB (Figure 4) as well as ELISA, FC, and protein purification.Myc tag monoclonal antibody for protein detection and immunoprecipitation studiesThe Myc tag, derived from the c-Myc gene product, is an epitope that can be used to detect recombinant proteins. The Myc tag can be fused A IP IP BControl IgG Anti-Myc Control IgG Anti-MycInput (Cat. No. 31903)(Cat. No. MA1-21316) Input (Cat. No. 31903)(Cat. No. MA1-21316)1 2 3 4 5 6 1 2 3 4 5 6Panel I 260 Panel III260160 160110 11080 MycNFkB p65 80 MycNFkB p6565 kDa 65 kDa60 IP: Anti-Myc60 IP: AntiNFkb p65 (Cat. No. MA1-21316) (Cat. No. 33-9900)50 WB: AntiNFkB p6550 WB: Anti-Myc (Cat. No. 33-9900) (Cat. No. MA1-21316)40 4030 302020151510Panel II Panel IVIP: Anti-Myc (Cat. No. MA1-21316) IP: AntiNFkb p65 WB: Anti-Myc (Cat. No. MA1-21316) (Cat. No. 33-9900)WB: AntiNFkb p65 (Cat. No. 33-9900)Figure 4. Immunoprecipitation and immunofluorescence analysis of Myc-tagged protein using a Myc tag monoclonal antibody. (A) Immunoprecipitation (IP) of Myc-tagged protein was performed using an Invitrogen Myc tag monoclonal antibody (Cat. No. MA1-21316; panels I, II) or Invitrogen NFkB p65 monoclonal antibody (Cat. No. 33-9900; panels III, IV). Lanes 1, 2: Total cell extract of HEK293E cells that were either untransfected (UT) or transfected (T) with a Myc-tagged NFkB p65 construct (7% of input shown). Lanes 3, 4: IP of UT and T HEK293E cell lysates with Invitrogen mouse IgG isotype control (Cat. No. 31903). Lanes 5, 6: IP of UT and T HEK293E cell lysates with 5 g of Myc tag monoclonal antibody (panels I, II) or NFkB p65 monoclonal antibody (panels III, IV). IPs were analyzed by western blot. Myc-tagged NFkB p65 was detected at ~65 kDa by probing the blot with NFkB p65 monoclonal antibody (panels I, IV) or Myc tag monoclonal antibody (1:1,000 dilution; panels II, III) and detected by chemiluminescence with Invitrogen Recombinant Protein G-HRP (Cat. No. 10-1223; 1:500 dilution) using the Invitrogen iBright FL 1500 Imaging System (Cat. No. A44115). Chemiluminescence detection was performed using Thermo Scientific Pierce ECL Western Blotting Substrate (Cat. No. 32106). (B) Panels AD: Immunofluorescence analysis of Myc-tagged protein was performed using 70% confluent HEK293 cells transfected with a Myc-tagged histone H3 construct. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 surfactant for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with Myc tag monoclonal antibody at 1:500 dilution and Invitrogen histone H3 recombinant polyclonal antibody (Cat. No. 711055) at 1:200 dilution in 0.1% BSA and incubated at 4C overnight. They were then labeled with Invitrogen goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 647 conjugate (Cat. No. A32728) and Invitrogen goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor Plus 488 conjugate (Cat. No. A32731) at a dilution of 1:2,000 for 45 minutes at room temperature. Cells were then mounted in Invitrogen ProLong Diamond Antifade Mountant with DAPI (Cat. No. P36962). Panel A (nuclei: red) shows Myc tag. Panel B (nuclei: green) shows histone H3. Panel C (nuclei: blue) shows cells stained with DAPI. Panel D is a merged image showing co-localization of nuclear signals in transfected cells (panels A, B, and C combined). Panels E and F show untransfected HEK293 cells mounted with ProLong Diamond Antifade Mountant with DAPI and Alexa Fluor Plus 488 goat anti-rabbit secondary antibody. Cells in Panel E were stained with the same histone primary antibody used in Panels A-D, while the cells shown in Panel F were not stained with the primary antibody.View all Invitrogen Myc tag antibodies at thermofisher.com/antibodiesContents Inside the Cell//Issue No. 11 17'