rabbit polyclonal anti-flag (60–031)  (BioAcademia)

Journal: Scientific Reports

Article Title: G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

doi: 10.1038/srep43480

Figure Lengend Snippet: ( a ) Western blot and immunoprecipitation (IP) analysis of FLAG-HA-Emerald GFP (FHG) tagged with C-terminal G196-tag in HeLa cells using mAb G196. HeLa cells were transfected with FHG/pcDNA3 or FHG-G196/pcDNA3. After incubation for two days, the cells were lysed and subjected to Western blotting with anti-FLAG (M2) or G196 mAbs ( left panel ). These lysates were immunoprecipitated with anti-FLAG (M2) (lanes 7, 8) or G196 (lanes 9, 10) mAbs and subjected to Western blotting with anti-FLAG polyclonal antibody ( right panel ). ( b ) Immunofluorescence of GFP- and G196-tagged nuclear protein in HeLa cells using mAb G196. HeLa cells were transfected with FHG-G196-NAC1/pcDNA3. After incubation for two days, the cells were fixed and incubated with mAb G196 and anti-GFP polyclonal Ab, then stained with Alexa-594 anti-mouse and Alexa-488 anti-rabbit secondary antibodies. ( c ) Western blot and chromatin immunoprecipitation (ChIP) analysis of Atf1-G196-GFP in fission yeast using mAb G196. A DNA fragment containing the transcription factor atf1 gene fused with the G196 epitope- GFP gene was replaced with the genomic atf1 gene. Protein extracts were separated by SDS-PAGE and detected with mAb G196 and anti-GFP polyclonal antibody ( left panel ). GFP-Iws1 expressing cells were used as a negative control. Asterisk indicates non-specific band also detected using the second Ab alone. Schematic representation of the tdh1 locus ( right upper panel ). The promoter of the tdh1 gene harbors a CRE consensus site. ChIP-qPCR analysis of the Arf1-G196-GFP level in the indicated regions ( right lower panel ). ( d ) Immunofluorescence of GFP- and G196-tagged nuclear protein in fission yeast cells using mAb G196. Atf1-G196-GFP expressing cells were fixed and incubated with mAb G196 and anti-GFP polyclonal Ab, then stained with Alexa-594 anti-mouse and Alexa-488 anti-rabbit secondary antibodies.

Article Snippet: The following commercial antibodies were used: mouse monoclonal anti-FLAG (M2, Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal anti-FLAG (60–031, BioAcademia, Osaka, Japan) and anti-GFP (60–011, BioAcademia); HRP-conjugated goat F(ab’) 2 anti-mouse (710–1332, Rockland Immunochemicals, Limerick, ME, USA) and goat anti-rabbit IgG (111–035–003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA); Alexa 488-conjugated goat anti-rabbit IgG(H+L) (A-11034, ThermoFisher Scientific, Waltham, MA, USA) and Alexa 594-conjugated goat anti-mouse IgG(H+L) (A-11032, ThermoFisher Scientific).

Techniques: Western Blot, Immunoprecipitation, Transfection, Incubation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, SDS Page, Expressing, Negative Control, ChIP-qPCR