Mouse Anti-G196 Tag Recombinant Antibody (clone G196) (CAT#: FAMAB-1349CQ)

Figure 1 Western blot analysis using mAb G196.

Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2-212). BamHI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines).

Figure 2 Isothermal titration calorimetry binding profile of G196 IgG Fab with the representative synthetic peptide GSDLVPRGS.

The top panel shows a typical calorimetric titration of 25 μM G196 IgG Fab with synthetic peptide at 25 °C. The bottom panel shows the integrated curve showing the experimentally obtained (◾) points and the best fit (−). The best fit to the data yielded n = 1.2 sites, Kd = 1.25 nM (±0.77), ΔH = −15.35 kcal/mol, and TΔS = −3.19 kcal/mol.

Figure 3 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.

Figure 4 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).

Figure 5 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.

Figure 6 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).