SDS–polyacrylamide gel electrophoresis and Western blotting

1. Place cells on ice and transfer to 1.5mL tube or other tube (10mL/15mL etc) depending on volume 

1. Pellet cells @ 4°C, 1400rpm, remove supernatant

1. Wash cells once in 1mL cold PBS in 1.5mL tube

1. Pellet cells @ 4°C, 1400rpm, remove supernatant

1. Whole cell lysis: resuspend cells in 1X Laemmli lysis buffer [60 mM tris-HCl (pH 6.8), 10% (v/v) glycerol, and 2% (w/v) SDS] – volume depends on cell pellet size, add enough that entire pellet is resuspended well

1. Incubate at 95°C for 10 minutes

1. Centrifuge @ room temperature, top speed, 1 minute

1. Determine protein concentration using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23225) as per manufacturer’s instructions

1. Prepare samples for SDS-PAGE equivalent to equal protein concentration (1 – 5mg/mL) with 2X sample loading buffer [120 mM Tris-HCl (pH 6.8), 20% (v/v) glycerol, 4% (w/v) SDS, 71.5 mM b-mercaptoethanol, and bromophenol blue].

Proceed to SDS-PAGE or store lysates at -20°C

1. SDS-PAGE: separate whole cell lysates using the Mini PROTEAN TGX 4 to 15% gradient gels (Bio-Rad, Hercules,CA, USA, 4561086) 

Running buffer: 25 mM tris, 192 mM glycine, and 0.1% (w/v) SDS in ultrapure H₂O

Voltage: 80 – 150V, room temperature

1. Wet-transfer of protein from SDS-PAGE gel to Immobilon-P (IPVH00010) or Immobilon-FL (IPFL00010) polyvinylidene fluoride membranes (Merck, Kenilworth, NJ, USA) using Bio-Rad Mini Trans-Blot^® Electrophoretic Transfer Cell system (Bio-Rad, Hercules,CA, USA, 1703930)

Transfer buffer: 25 mM Tris, 192 mM glycine, and 5% (v/v) methanol in ultrapure H₂O

Conditions:  4°C, 2 hours; 200 mA

1. Block membranes on roller @ room temperature for 30 minutes - 1 hour (or overnight @ 4°C)

Immobilon-P: tris-buffered saline (TBS) supplemented with 0.1% (v/v) Tween-20 (Sigma-Aldrich, P9416) and 5% (w/v) skim milk powder (Coles brand, purchased from supermarket)

Immobilon-FL: Odyssey blocking buffer (Li-COR, Lincoln, NE, USA, P/N 927-40000)

1. Incubate membranes overnight on roller @ 4°C with primary antibody

See attached table for antibody dilutions

Immobilon-P: dilute antibodies in TBS supplemented with 0.1% (v/v) Tween-20 and 5% (w/v) skim milk powder

Immobilon-FL: dilute antibodies in Odyssey blocking buffer supplemented with 0.2% Tween-20

1. Wash membranes @ room temperature 3x with TBS supplemented with 0.1% (v/v) Tween-20

1. Incubate membranes on roller for 1 hour @ room-temperature with secondary antibodies

See attached table for antibody dilutions

Immobilon-P: horseradish peroxidase (HRP)–conjugated secondary antibodies diluted in TBS supplemented with 0.1% (v/v) Tween-20 and 5% (w/v) skim milk powder

Immobilon-FL: IRDye-conjugated secondary antibodies diluted in Odyssey blocking buffer supplemented with 0.2% Tween-20 and 0.02% SDS

1. Wash membranes @ room temperature 3 – 5x with TBS supplemented with 0.1% (v/v) Tween-20 and 1x with TBS

1. Image development and visualization

Immobilon-P and HRP-conjugated secondary antibody: visualize using Amersham ECL Plus (GE Healthcare, Chicago, IL, USA, RPN2132) and Super RX film (Fujifilm, Tokyo, Japan, 03G01). Quantify using ImageJ software (version 1.46r).

Immobilon-FL and IRDye-conjugated secondary antibody: visualize using Odyssey CLx machine (settings: 84mM, medium) and Image Studio software (Li-COR)