ROBO1 Knockout A549 Cell Lysate

Western Blot: ROBO1 Knockout A549 Cell Lysate [NBP3-18631] - 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. Lane 9: MCF-7 WCL. Load: 35ug lysate/lane. Primary Antibody [Blot A] Anti-ROBO1 ~181kDa; [Blot B] stripped and re-probed with Anti-Tubulin ~50kDa; at 1ug/mL overnight at 2-8C. Secondary Antibody: Goat Anti-Rabbit IgG HRP at 1:70,000 for 30min at RT. Block: TTBS/Casein at RT. No detection of expected band at ~181kDa is observed in ROBO1 knockout A549 when compared with unmodified A549 cell lysates by Western blot.

Western Blot of ROBO1 Knockout A549 Cell Lysate. Lane 1: Opal Prestained MW Marker

• Reactivity: Hu
• Applications: WB, ELISA, IP, PAGE, ChIP

ROBO1 Knockout A549 Cell Lysate Summary

• Description: 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 uM Aprotinin, 5 uM Bestatin, 1.5 uM E-64, 2 uM Leupeptin Hemisulfate, 1 uM 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.
  
  Clone 15
  Lysate Fractionation: Whole Cell Lysate
  Lysate Stimulation: Not Stimulated
  Culture Type: Tissue Culture
• Preparation Method: 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 uM Aprotinin, 5 uM Bestatin, 1.5 uM E-64, 2 uM Leupeptin Hemisulfate, 1 uM 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.
• Gene: ROBO1
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • Chromatin Immunoprecipitation (ChIP)
  • ELISA
  • Immunoprecipitation
  • SDS-Page
  • Western Blot
• Application Notes: This product 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.

Packaging, Storage & Formulations

• Storage: Store at -70C. Avoid freeze-thaw cycles.
• Buffer: 1X RIPA Buffer with HALT Protease and Phosphatase Inhibitors
• Preservative: No Preservative
• Purity: Multi-step

Lysate Details for Array

• Type: Knockout A549 Cell
• Life Stage: Adult
• Subcellular Fraction: Whole

Alternate Names for ROBO1 Knockout A549 Cell Lysate

• DUTT1
• DUTT1Drosophila) homolog 1
• H-Robo-1
• MGC131599
• MGC133277
• ROBO1
• roundabout, axon guidance receptor, homolog 1 (Drosophila)

Background

Bilateral symmetric nervous systems have special midline structures that establish a partition between the two mirror image halves. Some axons project toward and across the midline in response to long-range chemoattractants emanating from the midline. In Drosophila, the roundabout gene, a member of the immunoglobulin gene superfamily, encodes an integral membrane protein that is both an axon guidance receptor and a cell adhesion receptor. This receptor is involved in the decision by axons to cross the central nervous system midline. The protein encoded by this gene is structurally similar to the Drosophila roundabout protein. Two transcript variants encoding different isoforms have been found for this gene.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Bioinformatics

• Gene Symbol: ROBO1
• Uniprot:
  • Q9Y6N7()
  • Q9Y6N7()
  • Q9Y6N7()
  • Q9Y6N7()