Mouse anti 6x his tag monoclonal antibody

Expi293F™ cells were maintained following the manufacturer’s instructions under standard culture conditions of 8% CO₂ and 37°C on an orbital oscillation platform (100–120 rpm) using Expi293 Expression Medium (Gibco #A1435102). A mouse anti-6X His-tag^® monoclonal antibody ([HIS.H8], Abcam 18184) and Peroxidase Affinity Pure Goat Anti-Mouse IgG (H + L) (Jackson Immuno Research # 115-035-003) was used for ELISA confirmation of the three recombinant proteins and western blot analyses. Anti-Human IgG (Fc specific)–peroxidase antibody produced in goat (Sigma, #A0170) was used for detection of SARS-CoV-2 IgG-specific antibodies present in human sera. Bovine serum albumin was used to block ELISA plates (Sigma, A9647-100G). Opti-MEM^® (1×) reduced serum medium for transfection experiments. Non-fat Omniblock skimmed milk (#AB101009, Americanbio) was used for blocking ELISA and western blot. Microtiter plates (Immunolon 4 HBX, Ultra-high binding polystyrene microtiter plates) for ELISA. A reference negative control serum (Accurun^® 810 Multi-Marker, 2017-11-11, Lot: 10087801, Seracare, United States) was used as a negative control.

The immunodetection was carried out according to previous reports^{38 (link)}. Briefly, nitrocellulose membranes were blocked in 5% (w/v) milk in 1X Tris buffered saline-Tween (TBST) for 1 h at room temperature. Membranes were then washed in TBST and incubated at room temperature overnight with the MUC2 rabbit polyclonal antiserum, MAN-2I, the MUC5AC rabbit polyclonal antiserum, MAN-5ACI, the MUC5B monoclonal antibody, EUMUC5B, and the mouse anti-6X His-tag monoclonal antibody [Cat# ab15149, RRID:AB_301694; Abcam, MA, USA] (for His-tagged MUC5B protein domains, NT5B and CT5B) all at a dilution of 1:2,000 in 1X TBST. Blots were then washed and incubated with fluorophore-conjugated secondary antibody [IRDye 800 goat anti-mouse (Cat# 926-32210, RRID:AB_621842) or IRDye 800 goat anti-rabbit (Cat# 926-32211, RRID:AB_621843); LI-COR Biosciences, Cambridge, UK] at a dilution of 1:25,000 in TBST for 60 min. Finally, each blot was imaged using a LI- COR Odyssey® CLx Infrared Imaging System.

Proteins were extracted from lung tissues or cells in lysis buffer (Thermo Fisher Scientific) using protease and phosphatase inhibitor cocktail (Roche Diagnostics), followed by centrifugation. Western blotting was performed as described previously^{36 (link)}. For each experiment, equal quantities of total protein were resolved by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The blots were subsequently blocked in 5% skimmed milk and incubated for 24 h at 4 °C with the following primary antibodies: mouse anti-6X His tag monoclonal antibody (1:1000; Abcam), rabbit anti-FASN polyclonal antibodies (1:500; Abcam, Cambridge), rabbit anti-caspase-3 (1:500; Cell Signaling Technology), rabbit anti-cleaved caspase-3 (1:500; Cell Signaling Technology), rabbit anti-PINK1 (1:500; Proteintech) and anti-β-actin monoclonal antibody (1:5000; Sigma-Aldrich). After washing several times with TBS containing Tween (TBST), the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG secondary antibody (GenDEPOT). The membranes were analyzed by chemiluminescence (Thermo Fisher Scientific and Bio-Rad, Berkeley, CA, USA) using a ChemiDoc™ Touch Imaging System (Bio-Rad).