Anti GFP  

Immunofluorescence Microscopy of chicken anti-GFP antibody. Tissue: KruppleGAL4 driver line in Drosophila eye disc. Fixation: 0.5% PFA. Antigen retrieval: not required. Primary antibody: anti-GFP antibody diluted 1:500 for 2 hr at RT. Secondary antibody: Alexa？488 conjugated anti-Chicken IgG at 1:300 for 1 hr at RT. Blocking: 5% NGS in PBS with 0.1% Triton X-100 for 15 min. Staining: recombinant tau-myc-GFP protein as green fluorescent signal.

Western Blot of anti-GFP antibody. Lane 1: mouse spleen lysate (p/n W10-000-T029). Lane 2: mouse spleen lysate spiked with 50 ng of wt GFP (p/n W10-000-T029/000-001-215). Load: 20 μg per lane. Primary antibody: GFP antibody at 2 μg/ml for 2 hr at room temperature. Secondary antibody: IRDye？800 Conjugated Affinity Purified anti-Chicken IgG [H&L] [Goat] MX10 (p/n 603-132-126) at 1:20,000 for 45 min at RT. Block: 5% BSA in PBS 2 hr at room temperature. Predicted/Observed size: 27 kDa for GFP epitope tag. Other band(s): none.

Western Blot of Anti-GFP (CHICKEN) antibody. Lane 1: MW. Lane 2: GFP (p/n 000-001-215). Load: 0.05 μg. Primary antibody: Anti-GFP (CHICKEN) antibody (p/n 600-901-215) was used at 1:1000 overnight at 4°C. Secondary antibody: Anti-Chicken IgG (GOAT) peroxidase conjugated antibody (p/n 603-103-126) secondary antibody was used at 1:40,000 in Blocking Buffer for Fluorescent Western Blotting (p/n MB-070). Block: 1% BSA-TTBS (p/n MB-013, diluted to 1X) 30 min at 20°C. Predicted/Observed size: 27 kDa for GFP. Other band(s): none.

Western Blot Results of Chicken Anti-GFP Antibody. Lane 1: Opal Prestained Molecular Weight Marker (p/n MB-210-0500). Lane 2: GFP (p/n 000-001-215) / HeLa WCL (p/n W09-000-364) [0.05μg/10μg]. Lane 3: GFP (p/n 000-001-215) / NIH3T3 WCL (p/n W10-000-371) [0.05μg/10μg]. Lane 4: GFP (p/n 000-001-215) / PC-12 WCL (p/n W120-001-GL9) [0.05μg/10μg]. Lane 5: HeLa WCL (p/n W09-000-364) [10μg]. Lane 6: NIH3T3 WCL (p/n W10-000-371) [10μg]. Lane 7: PC-12 WCL (p/n W120-001-GL9) [10μg]. Primary Antibody: Anti-GFP at 1.0μg/mL overnight at 2-8°C. Secondary Antibody: Goat Anti-Chicken IgG HRP (p/n 603-103-126) at 1:40,000 for 30 mins at RT. Block: MB-070 Buffer for 1hr at RT. Predicted MW: 27kda. Exposure: 2 seconds.

Western Blot of Chicken anti-GFP Antibody with Serums.Lane 1: Opal Prestained Molecular Weight Marker (p/n MB-210-0500). Lane 2: GFP/Human Serum (p/n 000-001-215/D314-05) [0.01/0.02μL] [+]. Lane 3: Human Serum (p/n D314-05) [0.02μL] [-]. Lane 4: GFP/Mouse Serum (p/n 000-001-215/D308-05) [0.01/0.02μL] [+]. Lane 5: Mouse Serum (p/n D308-05) [0.02μL] [-]. Lane 6: GFP/Rat Serum (p/n 000-001-215/D310-05) [0.01/0.02μL] [+]. Lane 7: Rat Serum (p/n D310-05) [0.02μL] [-]. Primary antibody: Anti-GFP antibody at 1.0ug/mL overnight at 2-8°C. Secondary antibody: Donkey Anti-Chicken IgG HRP secondary antibody (p/n 603-103-126) at 1:40,000 for 30 min at RT. Blocking Buffer: BlockOut Buffer (p/n MB-073) for 1hr at RT. Predicted/Observed size: ~27kDa for GFP.