ROBO-1 antibody

• Catalog Number: orb345475
• Category: Antibodies
• Description: ROBO-1 antibody
• Species/Host: Rabbit
• Clonality: Polyclonal
• Tested applications: ELISA, IF, IHC, IP, WB
• Reactivity: Human, Mouse
• Isotype: IgG
• Immunogen: This affinity-purified antibody was prepared from whole rabbit serum produced by repeated immunizations with a synthetic peptide corresponding to an C-Terminal region near amino acids 1625-1650 of Human ROBO-1.
• Concentration: 1.0 mg/mL
• Dilution range: ELISA: 1:30,000 - 1:60,000, IHC: 2 µg/ml to 10 µg/ml, IF: User Optimized, IP: User Optimized, WB: 1:500 - 1:3,000
• Form/Appearance: Liquid (sterile filtered)
• Purity: This affinity purified antibody is directed against human ROBO-1 protein. The product was affinity purified from monospecific antiserum by immunoaffinity purification. A BLAST analysis was used to suggest reactivity with this protein from human, mouse, rat and dog sources based on 100% homology for the immunogen sequence. Cross reactivity will occur with all isoforms of ROBO-1. Cross reactivity with ROBO-1 homologues from other sources has not been determined.
• Conjugation: Unconjugated
• UniProt ID: Q2M1J3
• NCBI: AAI12337.1
• Storage: Store vial at -20° C prior to opening. Aliquot contents and freeze at -20° C or below for extended storage. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room temperature. This product is stable for several weeks at 4° C as an undiluted liquid. Dilute only prior to immediate use.
• Buffer/Preservatives: 0.01% (w/v) Sodium Azide
• Alternative names: rabbit anti-ROBO1 antibody, ROBO 1, ROBO-1, hROBO-
• Note: For research use only
• Application notes: This affinity purified antibody has been tested for use in ELISA, western blot, and immunohistochemistry. It may be suitable for immunofluorescence and IP. Specific conditions for reactivity should be optimized by the end user. Expect a band at ~181 kDa in size corresponding to ROBO-1 by western blotting in the appropriate cell lysate or extract.
• Expiration Date: 12 months from date of receipt.

1/50 staining mouse lung tissue sections (adult, frozen 100 µM 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.

Biorbyt'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.

ELISA results of purified Rabbit anti-Robo-1 Antibody tested against BSA-conjugated peptide of immunizing peptide. Each well was coated in duplicate with 0.1 µg of conjugate. The starting dilution of antibody was 5 µg/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) (p/n orb347654) and TMB ELISA Peroxidase Substrate (p/n orb348651).

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.

Western Blot of Rabbit anti-Robo-1 antibody. Lane M: Super Signal Molecular Weight Marker. Lane 1: HeLa WCL (p/n orb348668). Load: 30 µg lysate. Primary antibody: Robo-1 antibody at 1:1000 for overnight at 4°C. Secondary antibody: Peroxidase rabbit secondary antibody (p/n orb347654) at 1:40000 for 30 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting (p/n orb348637) 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 (p/n orb348669). Lane 2: HeLa Lysate (p/n orb348668). Lane 3: MCF-7 Lysate (p/n orb348664). Lane 4: Jurkat Lysate. Lane 5: A431 Lysate (p/n orb348665). Lane 6: A549 Lysate (p/n orb348675). Lane 7: LNCap Lysate (p/n orb348694). Lane 8: MOLT-4 Lysate (p/n orb348696). Lane 9: Ramos Lysate. Lane 10: Raji Lysate (p/n orb348672). Lane 11: A-172 Lysate (p/n orb348708). Lane 12: NIH/3T3 Lysate (p/n orb348714). Load: 35 µg per lane. Primary antibody: ROBO1 antibody at 1 ug/ml overnight at 4C. Secondary antibody: Peroxidase rabbit secondary antibody (p/n orb347654) at 1:30000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS for 30 min at RT. Predicted/Observed size: 181 kDa for ROBO1.

Western blot using Biorbyt's Affinity Purified anti-ROBO-1 antibody shows detection of a band at ~181 kDa corresponding to ROBO-1 present in mouse brain lysate (p/n orb348715) (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 1:1000. Reaction occurred 2h at room temperature followed by washes and reaction with a 1:10000 dilution of IRDye™800 conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature.