Veriblot for ip detection reagent

Cells were lyzed in 1x Laemmli sample buffer containing 60 mM Tris pH 6.8, 2% SDS and supplemented with 150 mM NaCl, 1 mM EDTA, 5% glycerol and 7 mM beta‐mercaptoethanol. Liver tissues or tumor nodules (~30 mg) were homogenized in 1 ml RIPA lysis buffer (50 mM Tris pH 8.0, 150 mM sodium chloride, 1% SDS, 1% Triton X‐100, 0.5% sodium deoxycholate and supplemented with protease inhibitor cocktail) using a Dounce tissue grinder and incubated on ice for 2 h. Tissue/cell lysates were centrifuged at 21,000 g for 20 min to remove cell debris. Equivalent amount of protein samples (80‐100 μg) were prepared and mixed with 2x Laemmli sample buffer before subjecting to SDS–PAGE using 15% or 10% acrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% BSA (bovine serum albumin)/PBST (containing 0.1 % Tween20) for 1 h at room temperature and then probed with the indicated primary antibodies overnight at 1:1,000 dilution in 5% BSA/PBST, followed by the appropriate HRP‐conjugated anti‐mouse/rabbit secondary antibodies (Bethyl Laboratories) or VeriBlot for IP Detection Reagent (Abcam, ab131366) for IP blots to minimize cross‐reactivity with the IgG bands. Six washes in PBST with 5 min incubation between washes were carried out before and after the second antibody incubation.

IP of GRP94 was performed using cell lysates or concentrated media of CHO cells grown in suspension batch cultures. The volume of the sample was adjusted to 500 μL with cell extraction buffer and incubated with 5 μL of the anti‐GRP94 polyclonal antibody (rabbit, ab227293; Abcam) for 2 hours on a rotary shaker at room temperature or overnight at 4°C. The sample was then incubated with 50 μL of Dynabeads magnetic protein G beads (Thermo Fisher Scientific) for 1 hour at room temperature. Magnetic protein G beads were pulled down by a magnet, washed three times with 1× PBS, and eluted with 25 μL of 50 mM glycine (pH = 2.8). For western blot, VeriBlot for IP detection reagent (HRP) (ab131366, Abcam) was used to minimize the interference of IP antibody.

Whole cell lysates were prepared from 10 mL of exponentially growing yeast in YEPD media using lysis buffer (50 mm Tris‐Cl, pH 7.5, 50 mm NaCl, 1% Triton X‐100, and 1 mm PMSF) and vortexing with glass beads. Fifty microgram of extract was incubated with 20 µL Protein A/G beads (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at 4 °C then centrifuged for 5 min to preclear the lysate. Lysates were then incubated with rabbit polyclonal antiserum to full length S. cerevisiae eEF1A (Kinzy Laboratory, Piscataway, NJ, USA) for 1 h at 4 °C, and the complex was precipitated by incubation with Protein A/G beads (1 h, 4 °C). Control reactions contained 10 µg of mouse IgG antibody (Millipore, Burlington, MA, USA). Immunoprecipitates were washed four times with lysis buffer, eluted using Laemmli buffer, and separated by SDS/PAGE. Membranes were blotted with rabbit polyclonal antiserum to full length S. cerevisiae eEF1Bα (Kinzy Laboratory) (1 : 5000) or eEF1A (1 : 10 000), and the secondary antibody (1 : 2000) was VeriBlot for IP Detection Reagent (Abcam, Cambridge, UK).