ROBO1 Knockout A549 Cell Lysate

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