Western blot: Is p28 (nsp1) expressed in transfected cells?

Reagents

• PBS (Phosphate buffered saline)
• Triple-detergent lysis buffer (50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.1% SDS; 1% NP-40; 0.5% sodium deoxycholate; 1 mM EDTA
• Protease inhibitor cocktail
• Primary monoclonal mouse anti-HA antibody
• Primary polyclonal mouse antiactin antibody
• Secondary HRP conjugated goat anti-mouse IgG
• 3,3′-Diaminobenzidine (DAB)
• Kaleidoscope protein ladder (BIO-RAD)

Procedure

Harvest cells

1. Wash cell monolayer with phosphate-buffered saline
2. Lyse with triple-detergent lysis buffer containing a protease inhibitor cocktail
3. Collect the lysate and incubate over ice for 30 minutes
4. Centrifuge at 14.000 x g for 10 minutes at 4 degrees Celcius after which the proteins are in the supernant

SDS-PAGE

1. Performed as described in the SOP of the HZ University of Applied Sciences
2. Use a 14% polyacrylamide gel for separation (calculated protein weight with ExPASy tool and 14% is recommended by ThermoScientific

Western blot

1. Performed as described in the SOP of the HZ University of Applied Sciences
2. Use of primary monoclonal mouse anti-HA antibody to detect recombinant
3. Use of primary polyclonal mouse antiactin antibody to use as control
4. Use of secondary HRP conjugated goat anti-mouse IgG to detect primary antibodies
5. Use of DAB as conjugate to the HRP to make the bands visible