Anti-NAIP antibody (ab2549)

Western blot - NAIP antibody (ab2549)

All lanes : Anti-NAIP antibody (ab2549) at 1/1000 dilution

Lane 1 : Lysate prepared from transfected PC3 cells.
Lane 2 : Lysate prepared from non-transfected PC3 cells.
Lane 3 : Lysate prepared from transfected HeLa cells.
Lane 4 : Lysate prepared from non-transfected HeLa cells.
Lane 5 : Lysate prepared from non-transfected NTera2D1 cells.

Secondary
secondary antibody at 1/100000 dilution

Observed band size : 160 kDa (why is the actual band size different from the predicted?)
Additional bands at : 105 kDa (possible isoform),115 kDa,130 kDa (possible isoform). We are unsure as to the identity of these extra bands.

Detection of novel NAIP protein isoforms.

Cells were grown in 10 cm dishes. The human PC3, NTera2D1, and HeLa cell lines were selected to screen for NAIP proteins based on preliminary RT-PCR findings. Cells transfected with the expression vector encoding the Alu-derived NAIP ORF or untransfected controls were harvested by either scraping or trypsinization following two washes with cold PBS. Cell pellets were obtained by centrifugation and resuspended in RIPA (150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris, pH8) and NP40 (150 mM NaCl; 1% NP-40; 50 mM Tris, pH8) lysis buffers supplemented with a protease inhibitor cocktail, and subsequently quantified using the Qubit Fluorometer. RIPA provided clearer results for the NAIP-specific antibody. Bi-phased gels containing TEMED and APS (4% stacking, 9% separating) were used to resolve total cellular protein in electrophoresis running buffer (10×: 25 mM Tris; 192 mM glycine; 0.1% SDS). Subsequently, separated proteins were transferred using a Hoefer TE 22 tank transfer unit onto Immobilon-P PVDF membrane in fresh transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, 0.1% SDS). To assess NAIP protein isoforms in primary human tissues an IMB-103-50 Instablot membrane was used. Blocking of all membranes was performed in 5% reconstituted skimmed milk powder under constant agitation at 4° overnight. The following morning, blocking solution was replaced and fresh primary antibody was applied at 1/1000 dilution for one hour at room temperature under constant agitation. Washes were carried out with TBS-T (10×: 20 mM Tris;1.4 M NaCl;1% Tween-20) at room temperature in 5 minute intervals, no more than five times. Secondary antibody was diluted in fresh TBST and 1% blocking solution to a final concentration of 1/100,000, and incubated for one hour at room temperature under constant agitation.

Image from Dr MT Romanish et al, PLoS One. 2009 Jun 2;4(6):e5761, Fig 5.