Western lightning plus ecl enhanced chemiluminescence detection kit

Cell lysates were run on a non-reducing SDS-PAGE gel, and the protein was transferred to a nitrocellulose membrane. The membrane was then gently rocked in in TBST (0.1% Tween-20 in Tris-buffered-saline) blocking buffer with 5% (w/v) non-fat milk powder at room temperature for 1.5 hr. The blocked membrane was cut and then incubated in TBST with 1% non-fat milk powder containing either GADPH-HRP conjugate antibody (Cell Signaling; 1:10,000 dilution), CD19 SJ25 antibody (Cell Signaling; 1:500 dilution) or CD81 5A6 antibody (Recombinant, 0.4 mg/mL; 1:100 dilution). Antibody incubations were performed overnight at 4°C with shaking. The CD19 blot was incubated with Donkey anti Rabbit IgG (H+L) HRP Conjugate (Thermo Fischer) diluted 1:5000 in TBST containing 1% non-fat milk powder. The CD81 blot was incubated with Rabbit Anti-Human IgG H and L HRP Conjugate (Abcam) diluted 1:5000 in TBST with 1% non-fat milk powder. Secondary antibody incubations were performed for 1 hr at room temperature with shaking. Prior to chemiluminescent detection, blots were washed with TBST three times for 10 min each time. Western blots were developed with Western Lightning Plus-ECL, Enhanced Chemiluminescence Detection Kit (PerkinElmer). Densitometry was performed in ImageJ. CD19 and CD81 levels were normalized to GAPDH.