Anti-ROBO-1 Antibody

ELISA results of purified Rabbit anti-Robo-1 Antibody tested against BSA-conjugated peptide of immunizing peptide. Each well was coated in duplicate with 0.1µg of conjugate. The starting dilution of antibody was 5μg/ml and the X-axis represents the Log10 of a 3-fold dilution. This titration is a 4-parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using 3% fish gel, Goat anti-Rabbit IgG Antibody Peroxidase Conjugated (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) and TMB ELISA Peroxidase Substrate .

Western Blot of Rabbit anti-Robo-1 antibody. Lane M: Super Signal Molecular Weight Marker. Lane 1: HeLa WCL . Load: 30 µg lysate. Primary antibody: Robo-1 antibody at 1:1,000 for overnight at 4°C. Secondary antibody: Peroxidase rabbit secondary antibody at 1:40,000 for 30 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting for 30 min at RT. Predicted/Observed size: 181 kDa, 181 kDa for Robo-1. Other band(s): lower bands not identified.

Western Blot of Rabbit anti-ROBO1 antibody. Marker: Opal Pre-stained ladder . Lane 1: HEK293 lysate . Lane 2: HeLa Lysate . Lane 3: MCF-7 Lysate . Lane 4: Jurkat Lysate . Lane 5: A431 Lysate . Lane 6: A549 Lysate . Lane 7: LNCap Lysate . Lane 8: MOLT-4 Lysate . Lane 9: Ramos Lysate . Lane 10: Raji Lysate . Lane 11: A-172 Lysate . Lane 12: NIH/3T3 Lysate . Load: 35 µg per lane. Primary antibody: ROBO1 antibody at 1ug/mL overnight at 4C. Secondary antibody: Peroxidase rabbit secondary antibody at 1:30,000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS for 30 min at RT. Predicted/Observed size: 181kDa for ROBO1.

Boster's Affinity Purified anti-ROBO1 antibody was used at a concentration of 5 µg/ml to detect ROBO1 in a variety of tissues including multi-human, multi-brain and multi-cancer slides. This image shows staining of human brain tissue. Tissue was formalin-fixed and paraffin embedded. Personal Communication, Tina Roush, LifeSpanBiosciences, Seattle, WA.

Western blot using Boster's Affinity Purified anti-ROBO-1 antibody shows detection of a band at ~181 kDa corresponding to ROBO-1 present in mouse brain lysate (arrowhead). Approximately 35 µg of lysate was separated by 4-8% SDS-PAGE and transferred onto nitrocellulose. After blocking the membrane was probed with the primary antibody diluted to 1:1,000. Reaction occurred 2h at room temperature followed by washes and reaction with a 1:10,000 dilution of IRDye™800 conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature. IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

1/50 staining mouse lung tissue sections (adult, frozen 100µm wholemount sections) by IHC-Fr. The tissue was paraformaldehyde fixed and permeabilized with triton x-100 before incubation with the antibody for 16 hours at 4°C.

Staining of ROBO1 in undifferentiated, immortalized human podocytes by Immunocytochemistry/ Immunofluorescence. Cells were fixed with 2% paraformaldehyde and 4% sucrose at room temperature for 10 minutes. The cells were then washed once with PBS, permeabilized with 0.3% Triton X-100 for 10 minutes and incubated with blocking solution (2% FCS, 2% BSA, 0.2% fish gelatin) for 30 minutes, before further incubation with primary Ab for 1 hour. An Alexa Fluor 488 goat anti-rabbit IgG secondary antibody was used at a dilution of 1/200. DAPI was used for nuclear counterstaining. Image from Lindenmeyer MT et al. Systematic Analysis of a Novel Human Renal Glomerulus-Enriched Gene Expression Dataset. PLoS One. 2010 July 12;5(7):e11545, Fig 5.