Human ROBO1 Knockout A549 Cell Lysate Antibody

• Catalog Number: orb1784559
• Category: Antibodies
• Description: Human ROBO1 Knockout A549 Cell Lysate Antibody
• Clone Number: Clone 15
• Tested applications: ChIP, ELISA, IP, SDS-PAGE, WB
• Concentration: 2.0 mg/mL
• Dilution range: ELISA: User Optimized, ChIP: User Optimized, IP: User Optimized, WB: User Optimized
• Form/Appearance: Liquid (sterile filtered)
• Purity: ROBO1 knockout A549 cells were grown in Dulbecco’s medium supplemented with 10% fetal bovine serum. Cells were washed with PBS and then incubated on ice in modified RIPA buffer, containing 150 mM sodium chloride, 50 mM Tris HCl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholic acid , 0.1% SDS and 0.01% (w/v) sodium azide to lyse the cells. Protein integrity was ensured using a cocktail of protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases (0.1 mM AEBSF HCl, 0.08 µM Aprotinin, 5 µM Bestatin, 1.5 µM E-64, 2 µM Leupeptin Hemisulfate, 1 µM Pepstatin A). Phosphatase inhibitors 1 mM NaF and 1 mM Na3VO4 were also added. Cell debris was removed by centrifugation. Protein concentration was determined by BCA using a commercially available kit. Protein concentration was adjusted to 2 mg/ml with modified 1X RIPA buffer. ROBO1 knockout A549 Clone 15 contains knockout deletions on all three copies of the ROBO1 gene in A549 cells. Each copy contains the same 23bp deletion induced by CRISPR/Cas 9. The deletion occurs in exon 2 and disrupts the sequence encoding amino acids 62 through 69, causing the downstream amino acid sequences to shift out of frame resulting in early stop codons. Validated by Sanger sequencing and Western blot.
• Conjugation: Unconjugated
• UniProt ID: Q9Y6N7
• Source: Human
• Biological Origin: Human
• Storage: Store vial at -70° C or COLDER. For extended storage, aliquot contents to minimize freeze/thaw cycles.
• Buffer/Preservatives: Preservative: None. Stabilizer: None. 1X RIPA Buffer with HALT Protease and Phosphatase Inhibitors
• Alternative names: rabbit anti-ROBO1 antibody, ROBO 1, ROBO-1, hROBO-
• Note: For research use only
• Application notes: Human ROBO1 Knockout A549 Cell Lysate has been tested by SDS-PAGE and western blot and is suitable for use in Western blot, ELISA, Immunoprecipitation and ChIP. No detection of expected band at ~181kDa is observed in ROBO1 knockout A549 when compared with unmodified A549 cell lysates by Western blot. Induction: None (Control)
• Expiration Date: 12 months from date of receipt.

Western Blot of Human ROBO Knockout A549 Cell Lysate. Lane 1: Opal Prestained MW Marker. Lane 2: A549 ROBO KO Clone 5. Lane 3: A549 ROBO KO Clone 15. Lane 4: A549 ROBO KO Clone 17. Lane 5: A549 ROBO KO Clone 18. Lane 6: A549 ROBO KO Clone 21. Lane 7: A549 ROBO1 KO Bulk. Lane 8: A549 WCL Parental (p/n orb348675). Lane 9: MCF-7 WCL (p/n orb348664). Load: 35 µg lysate/lane. Primary Antibody [Blot A] Anti-ROBO1 (p/n orb345475) ~181 kDa; [Blot B] stripped and re-probed with Anti-Tubulin (p/n orb344425) ~50 kDa; at 1 µg/mL overnight at 2-8°C. Secondary Antibody: Goat Anti-Rabbit IgG HRP at 1:70000 for 30 min at RT. Block: TTBS/Casein at RT. No detection of expected band at ~181 kDa is observed in ROBO1 knockout A549 when compared with unmodified A549 cell lysates by Western blot.